Process for producing biopterin

ABSTRACT

The present invention provides a transformed cell which is transformed by at least one gene of enzymes participating in biosynthesis of tetrahydrobiopterin and a process for the production of a biopterin compound using the same. In accordance with the present invention, the biopterin compound can be produced in large quantities in an industrial advantageous manner from less expensive materials.

TECHNICAL FIELD

[0001] The present invention relates to transformed cell into whichgenes of one or more enzyme(s) participating in biosynthesis oftetrahydrobiopterin are introduced and also to a process for theproduction of a biopterin compound using the same.

BACKGROUND ART

[0002] In the present specification, tetrahydrobiopterin(L-erythro-5,6,7,8-tetrahydrobiopterin; hereinafter, referred to asTHBP), 7,8-dihydrobiopterin (L-erythro-7,8-dihydrobiopterin;hereinafter, referred to as DHBP) or biopterin (hereinafter, referred toas BP) or a combination of any two or more of them is called a biopterincompound as a whole.

[0003] THBP is a biosubstance which is widely distributed in animals andwas revealed to be a coenzyme of phenylalanine hydroxylase in liver ofrat by Kaufman in 1963. After that, it was also revealed that THBP is acoenzyme which commonly acts on tyrosine hydroxylase and tryptophanehydroxylase and was revealed to play an important role participating inbiosynthesis of neurotransmitters. In recent years, its function as acoenzyme for nitrogen monoxide (NO) synthase was also found and has beenreceiving public attention as a coenzyme for biosynthetic enzymes forvarious biosubstances. It has ATTACHMENT A been known that, in humanbeing, defect of THBP results in a reduction of activity of theabove-mentioned amino acid hydroxylase causing an abnormally highphenylketonuria and abnormally high phenylalaninuria and THBP is used asa therapy for such inborn metabolism error diseases as sapropterinhydrochloride.

[0004] It has been known that, in animals, THBP is biosynthesized fromguanosine triphosphate (hereinafter, abbreviated as GTP) by a three-stepenzymatic reaction using GTP cyclohydrase I (hereinafter, abbreviated asGCH), 6-pyruvoyltetrahydropterin synthase (hereinafter, abbreviated asPTPS) and sepiapterin reductase (hereinafter, abbreviated as SPR) asshown in FIG. 1. In the meanwhile, other than the animals, THBP has beenreported to be present in fruit fly (Weisberg, E. P. and O'Donnell, J.M. J. Biol. Chem. 261:1453-1458, 1996), silkworm (Iino, T., Sawada, H.,Tsusue, M. and Takikawa, S.-I. Biochim. Biophys. Acta 1297:191-199,1996), eukaryotic microbes such as Euglena gracillis, Neurospora crassaand Pycomyces blakesleeanus (Maier, J., Ninnemann, H. Photochamistry andPhotobiology 61:43-53, 1995) and Nocardia species which are prokaryoticmicrobes (Son, J. K., Rosazza J. P. J. Bacteriol. 182:3644-3648, 2000).As such, although there are many living things in which the presence ofTHBP is reported, microbes are particularly greatly different fromanimals in view of evolution and, therefore, it is believed that THBP isnot a biosubstance which is commonly available in microbes as a whole.

[0005] THBP is a compound having three asymmetric carbons in a molecularand, accordingly, it is a compound where its chemical synthesis isdifficult and, with regard to its main synthetic methods, there has beenknown a method where L-erythro-biopterin (BP) is synthesized usingrhamnose or deoxyarabinose as a starting material and then it ischemically subjected to an asymmetric reduction to synthesize theproduct. However, those chemically synthesizing methods have manyreaction steps and, during the reaction steps, there are processeshaving low reaction yields. There are also difficulties that rhamnoseand deoxyarabinose which are starting materials are expensive and theyare hardly said to be advantageous manufacturing methods in terms ofoperation, yield, cost, and so on.

[0006] Under such circumstances, Kagamiyama, et al. tried the synthesisof THBP by an enzymatic reaction, purified each of the three enzymesparticipating in the THBP synthesis and by using the three enzymessucceeded in synthesizing the THBP in a reactor containing GTP andnicotinamide dinucleotide phosphate (reduced form) (hereinafter,abbreviated as NADPH) (Japanese Patent Laid-Open No. 82,888/1992).However, in obtaining 1 kg of THBP by this method, 3.12 kg of GTP and 92kg of NADPH are necessary. When the facts that those materials are veryexpensive and that operations including purification of three kinds ofenzymes are very troublesome are taken into consideration, industrialproduction of the biopterin compound using the said method is difficultin view of cost and operations.

[0007] On the other hand, Shiraishi, et al. found that enzymes of genusCandida (Candida noveiius, Candida rugosa, Candida robsta, etc.) andseveral filamentous fungi of genus Mucor (Mucor javanicus, Mucoralternns, Mucor subtilissmus, etc.) accumulate L-erythro-biopterin (BP)which is an oxidized product of THBP in a medium and reported a processfor the production of BP from the culture medium utilizing the aboveproperty (Japanese Patent Laid-Open No. 9,296/1986 and No. 33,990/1993).Since BP is able to be converted to THBP by a chemical reduction, amethod where BP is produced by microbes and then converted to THBP doesnot need expensive GTP and NADPH and, further, greatly reduces thesynthesizing steps and, accordingly, it is an industrially advantageousmanufacturing method in view of operations. However, in any of thestrains, the amount of BP produced thereby is as low as that BPproduction per liter of the culture medium is not more than 1 mg and,therefore, it is not an industrially advantageous manufacturing methodin view of yield and cost.

[0008] Accordingly, THBP which is useful as such has been hardly anobject of study because there has been no industrially advantageousmanufacturing method and, in addition, its usefulness has not beenalways sufficiently utilized because THBP has been hardly provided aspharmaceuticals, etc. in general. Further, although the biopterincompound such as DHBP and BP which are oxidized products of THBP whichwill be mentioned below have been expected to exhibit the same or evenbetter pharmacological actions, the actions have not been fullyinvestigated.

DISCLOSURE OF THE INVENTION

[0009] An object of the present invention is to provide a process for anindustrially advantageous production of the biopterin compound in largequantities from less expensive starting material using a geneticengineering means.

[0010] Another object of the present invention is to provide atransformed cell being able to participate in biosynthesis oftetrahydrobiopterin into which gene of one or more enzyme(s) isintroduced or to provide an expression vector having gene of one or moreenzyme(s) being able to participate in biosynthesis oftetrahydrobiopterin used for the preparation of such a transformed cell.

[0011] Still another object of the present invention is to provide amutant host cell such as a genetically recombined cell having a GTPsynthetic ability of not less than the GTP synthetic ability of cells ofa wild type or to provide a mutant host cell or a transformed cellhaving a GCH activity of not less than the GCH activity inherent to thecells of a wild type which is advantageous for production oftetrahydrobiopterin.

[0012] The present inventors have found that, when gene of enzymeparticipating in biosynthesis of THBP is introduced into host cell ofmicrobe or the like which can be cultured in large quantities, thepresent substance can be biosynthesized at a low cost and in largequantities. Thus, since GTP which is a starting material for the THBPbiosynthesis is a biosubstance commonly available in living things, ithas been found that, when genes encoding enzymes for THBP biosynthesisin animals or other living things are introduced into a host cell by agenetic recombination technique, the host cell which is inherentlyunable to produce THBP is now able to produce THBP whereupon the presentinventors have carried out an intensive investigation for preparation ofsuch transformed cells.

[0013] On the other hand however, there is a possibility that synthesisof THBP in large quantities may be harmful to the host cell and it isforecasted that production of THBP by this method will be difficult aswell. Thus, since THBP has a similar structure to folic acid because ofthe presence of a pteridine ring therein, it may be also regarded as akind of folic acid analogs. Actually, there is a report wheredihydrofolic acid reductase of Escherichia coli acts on7,8-dihydrobiopterin (DHBP) which is an oxidized product of THBP wherebyDHBP is converted to THBP (Watanabe, et al. Seikagaku, volume 53, no. 8,p. 1008 (1981)). Folic acid is a coenzyme participating in variouscarbon transition reactions in vivo and is a substance essential for thegrowth of cells and, therefore, when THBP is synthesized insignificantly large amounts in cells, it is forecasted that theenzymatic reaction in which folic acid is participated is inhibited andthat inhibition of growth of cells and succeeding death of the cells areresulted.

[0014] There are also reported that 7,8-dihydroneopterin produced from7,8-dihydroneopterin triphosphate which is an intermediate for the THBPsynthesis forms a radical (Oettl, K., et al. Biochem. Biophys. Res.Commun. 264:262-7, 1999) and that, when neopterin which is an oxidizedproduct thereof is present together with hydrogen peroxide or nitrousacid, cytotoxicity of hydrogen peroxide and nitrous acid is furtherpotentiated (Wede, I., et al. Free Radic. Res. 1999November;31(5):381-8). Accordingly, when THBP is produced in largequantities, 7,8-dihydroneopterin triphosphate as an intermediate isaccumulated and that is converted to 7,8-dihydroneopterin by phosphatasein the cells whereby there might be a possibility of giving injury tothe cells by radical formation or there might be a possibility ofshowing a cytotoxicity due to the coexistence of neopterin which isproduced by oxidation with hydrogen peroxide and nitrous acid which aregenerated in the cells.

[0015] In addition, there is a risk of possibility that synthesis ofTHBP in large quantities reduces the pooling of GTP which is a substrateand NADPH which is a coenzyme in the cells whereby inhibition of thegrowth is resulted. Further, there is a risk of possibility that, whenan enzyme which is necessary for biosynthesis of THBP is highlyexpressed in the cells, that might cause inhibition of cell growth.Furthermore, it is absolutely ambiguous whether the highly produced THBPin the cells surely transfers to a medium efficiently.

[0016] Taking the toxicity to the host cells as such into consideration,the present inventors introduced the gene of the enzyme participating inthe THBP synthesis into Escherichia coli and Saccharomyces yeast whichinherently have no the said synthesizing ability and investigated theproductivity of biopterin compound. As a result, it was found that thosetransformed cells prepared as such did not cause inhibition of growthbeing worried about but produced biopterin compound in large quantitiesand was able to efficiently transfer the said biopterin compound to theculture medium. As a result of further invention, it was found thatproduction of the biopterin compound by transformed cells into whichgene of enzyme participating in the THBP synthesis was introduced waspossible.

[0017] The present inventors have furthermore investigated to increasethe production of the biopterin compound where Escherichia coli was usedas an example. To begin with, since the starting substance for the THBPsynthesis is GTP, preparation of host cells having a high GTPsynthesizing ability than that of the cells of a wild type (hereinafter,abbreviated as highly GTP-synthetic host cells) was investigated.

[0018] It was found that, since GTP is a kind of purine compound, hostcells carrying mutation for enhanced GTP synthesis were able to beobtained when mutant strain with deregulated purine biosynthesis pathwaywas screened. There is a report that, in the case of Bacillus subtilis,such a regulatory mutant strain for the purine synthesis can be obtainedfrom mutants having a resistance to purine analogs such as 8-azaguanineand decoynine (refer, for example, to Ishii and Shiio, Agric. Biol.Chem. 36(9):1511-1522, 1972; Matsui, et al., Agric. Biol. Chem.43(8):1739-1744, 1979) although, in the case of Escherichia coli, it hasbeen ambiguous whether such purine analogs are effective for collectionof highly GTP-synthetic host cells.

[0019] The present inventors succeeded in obtaining highly GTP-syntheticcells using purine analogs such as 8-azaguanine and decoynine even inthe case of Escherichia coli. The present inventors further found awonderful fact that, when such a regulatory mutant strain for the purinesynthesis where the GTP synthetic ability is potentiated was used as ahost cell, productivity for the biopterin compound was about ten-fold ascompared with the case where a wild type was used as a host cell and,moreover, DHBP was produced in a significant amount as well.

[0020] It was further investigated as a method for preparing the highlyGTP-synthesizing host cells that guaBA gene which codes for IMPdehydrogenase and GMP synthase converting inosinic acid to guanylic acidis introduced into cell to increase the activity of the said bothenzymes. As a result, it was found that, when the guaBA gene wasintroduced into Escherichia coli which is the above-mentioned regulatorymutant strain for purine synthesis and such genetically recombinant cellis used as a host cell, production of the biopterin compound furtherincreased as compared with the use of the already-mentioned regulatorymutant strain for the purine synthesis.

[0021] The present inventors furthermore carried out an intensiveinvestigation for increasing the productivity of the biopterin compoundand, as a result, they found that, when gene for the enzymeparticipating in the THBP synthesis is introduced into host cell forplural times to increase the expressed amount, there was an improvementin the productivity of the biopterin compound. It was unexpectedly foundthat, especially when GCH gene which is an enzyme for catalyzing thefirst step reaction for the THBP synthesis is introduced into the hostcell for plural times to increase the expressed amount of GCH,productivity of the biopterin compound significantly increased.

[0022] On the basis of the above finding that productivity for thebiopterin compound significantly increases when GCH gene is introducedinto host cells for plural times, the use of GCH gene of Bacillussubtilis (may also be called mtrA gene) was investigated whereupon itwas unexpectedly found that the produced amount of the biopterincompound further increased.

[0023] As such, the present invention is able to provide an industriallyadvantageous process for producing the biopterin compound such as THBPand also to provide transformed cells and host cells which are usefulfor such a production.

[0024] As a result of further investigation, the present inventors haveachieved the present invention.

[0025] Thus, the present invention relates to:

[0026] (1) In a process for production of the biopterin compound by atransformed cell, a process for production of the biopterin compoundwhich is characterized in that (a) a host cell is transformed by atleast one gene of enzyme participating in biosynthesis oftetrahydrobiopterin, (b) the resulting transformed cell is cultured toproduce tetrahydrobiopterin, (c) the resulting tetrahydrobiopterin isoxidized if necessary and (d) one or more biopterin compound(s) selectedfrom the resulting tetrahydrobiopterin and dihydrobiopterin andbiopterin where the said tetrahydrobiopterin is oxidized is/arecollected;

[0027] (2) the process for production of the biopterin compoundmentioned in the above (1), wherein the biopterin compound in theculture broth or in a processed product thereof is oxidized andbiopterin is collected therefrom;

[0028] (3) the process for production of the biopterin compoundmentioned in the above (1) or (2), wherein the collecteddihydrobiopterin and/or biopterin are/is reduced to producetetrahydrobiopterin;

[0029] (4) the process for production of the biopterin compoundmentioned in the above (1) to (3), wherein the enzyme(s) participatingin the biosynthesis of tetrahydrobiopterin is/are one to three kind(s)of enzyme(s) selected from a group consisting of GTP cyclohydrase1,6-pyruvoyltetrahydropterin synthase and sepiapterin reductase;

[0030] (5) the process for production of the biopterin compoundmentioned in the above (1) to (3), wherein the transformation is carriedout by an expression vector having gene of 6-pyruvoyltetrahydropterinsynthase and gene of sepiapterin reductase;

[0031] (6) the process for production of the biopterin compoundmentioned in the above (5), wherein the host cell is a mutant cellhaving a GTP cyclohydrase I activity which is not less than the GTPcyclohydrase I activity inherent to the cell of a wild type;

[0032] (7) the process for production of the biopterin compoundmentioned in the above (4), wherein the GTP cyclohydrase I gene to beintroduced into the host cell is mtrA gene derived from Bacillussubtilis;

[0033] (8) the process for production of the biopterin compoundmentioned in the above (1) to (7), wherein the host cell is aprokaryotic cell;

[0034] (9) the process for production of the biopterin compoundmentioned in the above (8), wherein the prokaryote is Escherichia coli,Bacillus subtilis or Actinomyces;

[0035] (10) the process for production of the biopterin compoundmentioned in the above (1) to (7), wherein the host cell is aneukaryotic cell;

[0036] (11) the process for production of the biopterin compoundmentioned in the above (10), wherein the eukaryote is yeast orfilamentous fungi;

[0037] (12) the process for production of the biopterin compoundmentioned in the above (11), wherein the yeast is methanol assimilatingyeast or fission yeast;

[0038] (13) the process for production of the biopterin compoundmentioned in the above (11), wherein the yeast is Saccharomyces yeast;

[0039] (14) the process for production of the biopterin compoundmentioned in the above (1) to (13), wherein the host cell is a mutantcell having a GTP synthesizing ability of not less than the GTPsynthesizing ability of the cell of a wild type;

[0040] (15) the process for production of the biopterin compoundmentioned in the above (14), wherein the host cell is a mutant cellhaving a 8-azaguanine resistance of not less than the 8-azaguanineresistance of the cell of a wild type;

[0041] (16) the process for production of the biopterin compoundmentioned in the above (14) or (15), wherein the host cell is agenetically recombinant cell where guaBA gene coding for IMPdehydrogenase and GMP synthase is introduced thereinto;

[0042] (17) a transformed cell used for the production of the biopterincompound mentioned in the above (1) to (16) in which gene of an enzymeparticipating in biosynthesis of tetrahydrobiopterin is introduced intoa host cell;

[0043] (18) the transformed cell according to the above (17), whereinthe host cell is a mutant cell having a GTP synthesizing ability of notless than the GTP synthesizing ability of the cell of a wild type;

[0044] (19) the transformed cell according to the above (18), whereinthe host cell is a mutant cell having a 8-azaguanine resistance of notless than the 8-azaguanine resistance of the cell of a wild type;

[0045] (20) the transformed cell according to the above (18) or (19),wherein the host cell is a genetically recombinant cell where guaBA genecoding for IMP dehydrogenase and GMP synthase is introduced thereinto;

[0046] (21) the transformed cell according to the above (18), whereinthe host cell is a mutant cell having a GTP cyclohydrase I activitywhich is not less than the GTP cyclohydrase I activity inherent to thecell of a wild type and gene of 6-pyruvoyltetrahydropterin synthase andgene of sepiapterin reductase are introduced into the said host cell;

[0047] (22) the transformed cell according to the above (17)-(21),wherein the GTP cyclohydrase I gene to be introduced into the host cellis mtrA gene derived from Bacillus subtilis;

[0048] (23) the process for production of the biopterin compoundmentioned in the above (14) or (15), wherein a transformed cell whereguaBA gene coding for IMP dehydrogenase and GMP synthase is introducedinto the host cell is used; and

[0049] (24) a transformed cell mentioned in the above (18) or (19) whereguaBA gene coding for IMP dehydrogenase and GMP synthase is introducedinto a host cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1 shows a pathway for biosynthesis of tetrahydrobiopterin.

[0051]FIG. 2 shows a pathway for oxidation and decomposition oftetrahydrobiopterin.

[0052]FIG. 3 shows the steps for preparation of pSTV28-GCH. PCR (P1, P2)means a PCR using sense primer P1 and antisense primer P2. PCR (P3, P4)and PCR (P5, P6) have the similar meanings as well. Ap meansampicillin-resistant gene and Cm means chloramphenicol-resistant gene.trc means trc promoter and lac means lac promoter.

[0053]FIG. 4 shows the steps for preparation of pUC18-PTPS. PCR (P7, P8)means a PCR using sense primer P7 and antisense primer P8. PCR (P9, P10)and PCR (P11, P12) have the similar meanings as well. Ap meansampicillin-resistant gene. trc means trc promoter and lac means lacpromoter.

[0054]FIG. 5 shows the steps for preparation of pUC19-SPR. PCR (P13,P14) means a PCR using sense primer P13 and antisense primer P14. PCR(P15, P16) and PCR (P17, P18) have the similar meanings as well. Apmeans ampicillin-resistant gene. trc means trc promoter and lac meanslac promoter.

[0055]FIG. 6 shows the steps for preparation of pYES2-FOL2. PCR (P23,P24) means a PCR using sense primer P23 and antisense primer P24. Apmeans ampicillin-resistant gene and URA3 means a selective marker inyeast. PGALL means GALl promoter and CYCi TT means transcriptiontermination signal of CYCI gene.

[0056]FIG. 7 shows the steps for preparation of pSTV28-GPS. PCR (P19,P20) means a PCR using sense primer P19 and antisense primer P20. PCR(P21, P22) has the similar meaning as well. Ap meansampicillin-resistant gene and Cm means chloramphenicol-resistant gene.lac means lac promoter. Ori means origin of replication.

[0057]FIG. 8 shows the steps for preparations of pYES2-PTPS andpYES2-SPR. PCR (P25, P26) means a PCR using sense primer P25 andantisense primer P26. PCR (P27, P28) has the similar meaning as well. Apmeans ampicillin-resistant gene and URA3 means URA3 gene used as aselective marker in yeast. lac means lac promoter, PGAL1 means GALLpromoter and CYC1 TT means transcription termination signal of CYCIgene.

[0058]FIG. 9 shows the steps for preparations of pYES2-FPS. PCR (P29,P30) means a PCR using sense primer P29 and antisense primer P30. PCR(P31, P32) has the similar meaning as well. Ap meansampicillin-resistant gene and URA3 means a selective marker in yeast.PGAL1 means GALL promoter and CYCL TT means transcription terminationsignal of CYC1 gene.

[0059]FIG. 10 shows an amino acid sequence corresponding to a DNA basesequence of GCH cloned on pSTV28-GCH. The underlined part showsasequence derivedfrom pSTV28 to beaddedto an amino terminus of GCH.

[0060]FIG. 11 shows an amino acid sequence corresponding to a DNA basesequence of PTPS cloned on pUC18-PTPS. The underlined part shows asequence derived from pUC18 to be added to an amino terminus of PTPS.

[0061]FIG. 12 shows an amino acid sequence corresponding to a DNA basesequence of SPR cloned on pUC19-SPR. The underlined part shows asequence derived from pUC19 to be added to an amino terminus of SPR.

[0062]FIG. 13 shows the result of HPLC analysis of culture supernatantof biopterin compound producing Escherichia coli JM101/pSTV28-GPS by aC18 reversed column.

[0063]FIG. 14 shows growth curves of biopterin compound-productive yeast(FPS strain).

[0064]FIG. 15 shows the result of HPLC analysis of culture supernatantof biopterin compound producing yeast (FPS strain) by a C18 reversedcolumn.

[0065]FIG. 16 shows the result of TLC analysis of culture supernatant ofbiopterin compound producing yeast (FPS strain).

[0066]FIG. 17 shows the result of HPLC analysis of culture supernatantof biopterin compound producing Escherichia coli AG14/pSTV28-GPS by aC18 reversed column.

[0067]FIG. 18 shows the time course of BP and P production by E. coliAG14/pSTV28-GPS and the time course of OD660 of culture broth.

[0068]FIG. 19 shows the steps for preparation of pMW218-guaBA. PCR (P33,P34) means a PCR using sense primer P33 and antisense primer P34. Apmeans ampicillin-resistant gene and km means kanamycin-resistant gene.lac means lac promoter.

[0069]FIG. 20 shows the steps for preparation of pSTV28-MPS. PCR (P35,P36) means a PCR using sense primer P35 and antisense primer P36. PCR(P37, P38) has the similar meaning as well. Ap meansampicillin-resistant gene and km means kanamycin-resistant gene. Orimeans origin of replication.

[0070]FIG. 21 shows the time course of BP production by E. coliAG14/(pSTV28-GPS, pMW218-guaBA) and AG14/(pSTV28-MPS, pMW218-guaBA) andthe time course of OD660 of culture broth. In the drawing, ▪ showschanges with a lapse of time of OD660 for AG14/(pSTV28-GPS,pMW218-guaBA);  shows changes with a lapse of time of OD660 forAG14/(pSTV28-MPS, pMW218-guaBA); □ shows changes with a lapse of time ofproduced amount of BP for AG14/(pSTV28-GPS, pMW218-guaBA); and ∘ showschanges with a lapse of time of produced amount of BP forAG14/(pSTV28-MPS, pMW218-guaBA).

[0071]FIG. 22 shows the steps for preparation of pUC18SD. Ap meansampicillin-resistant gene. lac means lac promoter.

[0072]FIG. 23 shows the steps for preparation of pUC18AE. Ap meansampicillin-resistant gene. lac means lac promoter.

[0073]FIG. 24 shows the steps for preparation of pUC18AESDmtrA. PCR(P43, P44) means a PCR using sense primer P43 and antisense primer P44.PCR (P45, P46) and PCR (P45, P47) have the similar meanings as well. Apmeans ampicillin-resistant gene, mtrA means mtrA gene and SD means genecontaining SD sequence and DNA sequence coding for translationinitiation region. lac means lac promoter.

[0074]FIG. 25 shows the steps for preparation of pUC18SDPTPS. PCR (P50,P48) means a PCR using sense primer P50 and antisense primer P48. PCR(P50, P49) has the similar meaning as well. Ap meansampicillin-resistant gene, PTPS means PTPS gene and SD means genecontaining SD sequence and DNA sequence coding for translationinitiation region. lac means lac promoter.

[0075]FIG. 26 shows the steps for preparation of pUC18AESDSPR. PCR (P53,P51) means a PCR using sense primer P53 and antisense primer P51. PCR(P53, P52) has the similar meaning as well. Ap meansampicillin-resistant gene, SPR means SPR gene and SD means genecontaining SD sequence and DNA sequence coding for translationinitiation region. lac means lac promoter.

[0076]FIG. 27 shows the steps for preparation of pSL1180PS. Ap meansampicillin-resistant gene, PTPS means PTPS gene, SPR means SPR gene andSD means gene containing SD sequence and DNA sequence coding fortranslation initiation region. lac means lac promoter.

[0077]FIG. 28 shows the steps for preparation of pSL1180 MPS. Ap meansampicillin-resistant gene, PTPS means PTPS gene, SPR means SPR gene,mtrA means mtrA gene and SD means gene containing SD sequence and DNAsequence coding for translation initiation region. lac means lacpromoter.

[0078]FIG. 29 shows the steps for preparation of pDG148 MPS. Km meanskanamycin-resistant gene, Ap means ampicillin-resistant gene, PTPS meansPTPS gene, SPR means SPR gene and mtrA means mtrA gene. Pspac means spacpromoter, lacO means laco promoter and lacI means lacI gene. Ori meansorigin for replication.

[0079]FIG. 30 shows the steps for preparation of pDG148 MPSAI. Km meanskanamycin-resistant gene, Ap means ampicillin-resistant gene, PTPS meansPTPS gene, SPR means SPR gene and mtrA means mtrA gene. Pspac means spacpromoter, laco means laco promoter and lacI means lacI gene. Ori meansorigin for replication.

[0080]FIG. 31 shows amino acid sequence corresponding to DNA basesequence of mtrA cloned on pUC18AESDmtrA. The underlined part showsamino acid sequence derived from CcpA protein to be added to aminoterminus of mtrA (Fujita, et al., Microbiology, 140:6571-6580, 1998) andbase sequence corresponding thereto.

[0081]FIG. 32 shows amino acid sequence corresponding to DNA basesequence of PTPS cloned on pUC18SDPTPS. The underlined part shows aminoacid sequence derived from CcpA protein to be added to amino terminus ofPTPS (Fujita, et al., Microbiology, 140:6571-6580, 1998) and basesequence corresponding thereto.

[0082]FIG. 33 shows amino acid sequence corresponding to DNA basesequence of SPR cloned on pUC18AESDSPR. The underlined part shows aminoacid sequence derived from CcpA protein to be added to amino terminus ofSPR (Fujita, et al., Microbiology, 140:6571-6580, 1998) and basesequence corresponding thereto.

[0083]FIG. 34 shows HPLC analysis of culture supernatant of Bacillussubtilis strains 1A1/pDG148 MPS and 1A1/pDG148 MPSA I.

BEST MODE FOR CARRYING OUT THE INVENTION

[0084] With regard to the enzyme which is able to participate inbiosynthesis of tetrahydrobiopterin, there are exemplified enzymes suchas guanosine triphosphate cyclohydrase I (GCH),6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase(SPR) shown in FIG. 1.

[0085] With regard to the gene to be introduced into a host cell,anything may be used so far as it contains gene which codes for theabove-mentioned enzyme. To be more specific, not only DNA containing GCHgene derived from Escherichia coli as shown in FIG. 10 or GCH genederived from Bacillus subtilis as shown in FIG. 31, PTPS gene derivedfrom rat as shown in FIG. 11 and FIG. 32 and SPR gene derived from ratas shown in FIG. 12 and FIG. 33 but also DNA which hybridizes with thesaid DNA may be used. Examples of the DNA which is able to hybridizewith the said DNA are DNA containing a base sequence having a homologyof not less than about 80%, preferably not less than about 85%, morepreferably not less than about 90% and, most preferably, not less thanabout 95% to the base sequence shown in FIGS. 10-12 and FIGS. 31-33.

[0086] Hybridization may be carried out by a known method per se or amethod similar to that such as a method mentioned in “Molecular Cloning”2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989).When commercially available cDNA library or kit is used, a methodmentioned in the instructions attached thereto may be used. Morepreferably, hybridization may be carried out under a highly stringentcondition. An example of a highly stringent condition is a conditionwhere sodium concentration is about 19 to 40 mM or, preferably, about 19to 20 mM and temperature is about 50 to 70° C. or, preferably, about 60to 65° C. Especially, the case where sodium concentration is about 19 mMand temperature is about 65° C. is most preferred.

[0087] DNA upon introduction into a host cell may be any of genomic DNA,cDNA derived from cells or tissues and synthetic DNA. Vector which isused for introduction of gene may be any of bacteriophage, plasmid andcosmid. Further, cDNA may be directly amplified by a reversetranscriptase polymerase chain reaction (hereinafter, abbreviated asRT-PCR) using total RNA or mRNA fraction prepared from theabove-mentioned cells or tissues. Furthermore, that which ismanufactured by a synthetic method for oligonucleotide which is knownper se may be used. To be more specific, an example is a method wherechemical synthesis is carried out by a DNA synthesizer such as model 392(manufactured by Perkin Elmer) utilizing a phosphoramidite method.

[0088] More specifically, DNA containing an aimed gene to be introducedinto a host cell may, for example, be prepared as follows.

[0089] With regard to GCH which is an enzyme catalyzing the reaction ofthe first step in the tetrahydrobiopterin biosynthesis, it is present inmany host cells such as Escherichia coli, yeast cells, etc. in additionto animals as an enzyme participating in the folic acid synthesis and,therefore, DNA coding for GCH may be easily prepared, for example, by(1) a method where PCR is carried out using a synthetic DNA primerhaving a partial base sequence of the said DNA whereby DNA containingthe aimed gene is amplified from a gene library etc. or (2) a methodwhere selection is carried out by hybridization of an appropriate genelibrary with a thing where labeled with DNA fragment or synthetic DNAcoding for a part of or whole region of GCH (probe). Such methods may beappropriately used in the present invention and, between the two, theformer is preferred.

[0090] A method for hybridization may, for example, be carried out by amethod mentioned in “Molecular Cloning” 2nd edition (J. Sambrook, etal., Cold Spring Harbor Lab. Press, 1989), etc. When commerciallyavailable library or kit is used, a method mentioned in the instructionattached thereto may be used. DNA having a partial base sequence used asa probe for hybridization may also be manufactured according to asynthetic method for oligonucleotide which is known per se.

[0091] In the present invention, it is preferred to use GCH gene ofEscherichia coli (may also be called folE gene), GCH gene of yeast (mayalso be called FOL2 gene) or GCH gene of Bacillus subtilis (may also becalled mtrA gene) as a GCH gene and it is more preferred to use GCH geneof Bacillus subtilis.

[0092] In order to obtain the DNA coding for PTPS and SPR which carryout the reactions of the second step and the third step for thetetrahydrobiopterin biosynthesis, a method where mRNA is extracted fromliver of rat and then subjected to an RT-PCR is preferred. In extractingthe said mRNA, a method which is known per se may be used. Cells arepartially or completely destroyed using a surface-active agent such asNP-40, SDS, Triton X 100 or deoxycholic acid or using a physical meanssuch as homogenizer or freeze-melting and then mRNA is separated. Inorder to prevent the degradation of RNA by RNase during the extraction,it is preferred to add an RNase inhibitor such as heparin, polyvinylsulfate, bentonite, macaloid, diethyl pyrocarbonate or vanadium complexto the extract. With regard to the purification of mRNA containingpolyA, that may be carried out by a purifying method by affinity columnchromatography using poly U-Sepharose or the like where oligodT-cellulose or Sepharose 2B is a carrier or by a batch method using thepoly U-Sepharose or the like, fractionation by an SDG centrifugal methodor by an agarose electrophoretic method. cDNA is synthesized from mRNAfraction containing mRNA corresponding to PTPS and SPR obtained as such.With regard to a method for the synthesis of cDNA, methods which areknown per se may be used and there is exemplified a method where,firstly, a single stranded DNA complementary to mRNA is synthesized by areverse transcriptase in the presence of dATP, dGTP, dCTP and dTTP usingmRNA as a template and oligo dT as a primer, then the template mRNA isdigested and removed by treating with an alkali and, after that, doublestranded cDNA is synthesized by a reverse transcriptase or DNApolymerase using the said single stranded cDNA as a template.

[0093] In order to select the cDNA containing the gene of the enzymeparticipating in synthesis of THBP such as GCH, PTPS or SPR from thecDNA library prepared as such, a hybridization method where DNA fragmentcontaining the said enzyme gene is a probe is used. With regard to thehybridization method, there may be used the methods known per se such asthat mentioned hereinabove. In order to check whether the selected cDNAcodes for the enzyme participating in synthesis of THBP such as GCH,PTPS or SPR, it may be carried out in such a manner that cDNA isintegrated, for example, into vector which is reproducible inEscherichia coli or COS cells and the vector is introduced intoEscherichia coli or COS cells whereupon the enzyme is expressed.

[0094] The transformed cell in accordance with the present invention isusually such a thing that where the biopterin compound can be producedor productivity of the biopterin compound is improved by introduction ofgene of at least one enzyme participating in the above THBP synthesisinto host cell.

[0095] Accordingly, the host cell may be that which inherently has noproducing ability of the biopterin compound or has some ability forproducing the biopterin compound. Thus, among host cells, some of theminherently have any of 1 to 3 kind(s) of gene(s) among the enzymesparticipating in the above THBP synthesis and well express the said 1 to3 kind(s) of enzyme(s). In that case, gene(s) of such enzyme(s) may notbe introduced into the host cell so far as such enzyme(s) is/areconcerned. Of course, it is still possible to introduce it/them forenhancing the productivity of the biopterin compound. However, if thatis not the case or, in other words, gene(s) of the above-mentionedenzyme(s) is/are not present, it is usually necessary to introduce thegene of the said enzyme and, in case the said gene is present but theenzyme is not sufficiently expressed, it is preferred to introduce thegene of the said enzyme.

[0096] For example, the host cell may be prokaryotes such as bacteria ofgenus Escherichia, bacteria of genus Bacillus and bacteria of orderActinomyce tales or may be eukaryotes such as yeast, filamentous fungi,insect cell, insect, animal cell and plant cell.

[0097] A specific example of the bacteria of genus Escherichia is theso-called colon bacillus and its examples are Escherichia coli K12.DH1[Proc. Natl. Acad. Sci. USA, volume 60, 160 (1968)], JM101, JM103[Nucleic Acids Research, volume 9, 309 (1981)], JA221 [J. Mol. Biol.,volume 120, 517 (1978)], HB101 [J. Mol. Biol., volume 41, 459 (1969)]and C600 [Genetics, volume 39, 440 (1954)]. Among them, it is preferredto use Escherichia coli JM101 in the present invention.

[0098] An example of the bacteria of genus Bacillus is the so-called haybacillus and its examples are Bacillus subtilis 1A1 strain (trpC2)(Fujita, et al., Microbiology, 140:6571-6580, 1998) or MI114 [Gene,volume 24, 255 (1983)] and207-21 [Journal of Biochemistry, volume 95, 87(1984)]. In the present invention, it is particularly preferred to usethe former.

[0099] An example of bacteria of order Actinomycetales is the so-calledactinomyces and specific examples thereof are genus Streptomyces, etc.Examples of the microbe belonging to genus Streptomyces are Streptomyceslividans 3131, etc.

[0100] With regard to the yeast, it is preferred to use yeast of genusSaccharomyces, methanol assimilating yeast or fission yeast. An exampleof the methanol assimilating yeast is Pichia pastoris and an example ofthe fission yeast is Schizosaccharomyces pombe. Examples of the yeast ofgenus Saccharomyces are Saccharomyces cerevisiae KA31, Saccharomycescerevisiae AH22, AH22R-, NA87-11A, DKD-5D and 20B-12,Schizosaccharomyces pombe NCYC1913 and NCYC2036 and Pichia pastorisKM71. Among them, it is preferred to use yeast of genus Saccharomycesand particularly preferred to use Saccharomyces cerevisiae KA31 in thepresent invention.

[0101] Examples of the filamentous fungi are species belonging toAcremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,Neurospora, Penicillium, Thielavia, Tolypocladium and Trichoderma.

[0102] Examples of the insect cell are established cell derived fromlarva of Spodoptera frugiperda (Sf cell), MG1 cell derived from midgutof Trichoplusia ni, High Five TM cell derived from egg of TrichooplusianI, cell derived from Mamestrabrassicae and cell derived from Estigmenaacrea. Established cell derived from silkworm (Bombyx mori N; BmN cell),etc. may also be used. With regard to the above-mentioned Sf cell, theremay be exemplified Sf9 cell (ATCC CRL 1711), Sf21 cell (for those, cf.Vaughn, J. L., et al., in Vivo, volume 13, 213-217 (1977)), etc.

[0103] With regard to the insect, there may be exemplified larva ofsilkworm, etc. (Maeda, et al., Nature, volume 315, 592 (1985)).

[0104] With regard to the animal cell, there may be exemplified monkeycell COS-7, Vero cell, Chinese hamster cell CHO, dhfr gene-deficientChinese hamster cell CHO, mouse L cell, mouse AtT-20 cell, mouse myelomacell, rat GH3 cell, human FL cell, 293 cell, C127 cell, BALB/3T3 celland Sp-2 cell.

[0105] With regard to the plant cell, there may be exemplified tobaccocell and carrot cell.

[0106] With regard to the host cell in accordance with the presentinvention, there may be used a mutant cell having a GTP synthesizingability which is not less than the GTP synthetic ability of the wildtype cell of the above-mentioned host cell. The reason is that, sinceGTP which is a starting substance for the THBP synthesis is a kind ofpurine compound, supplying amount of GTP which is a starting substanceincreases when a regulatory mutant of purine synthesis pathway where theGTP synthesizing ability increases is used and, as a result, productionof the biopterin compound increases as well. In order to obtain aregulatory mutant of purine synthesis pathway, a known method may beused. For example, a method where incubation is carried out using amedium to which a purine analog is added and a mutant having aresistance to the purine analog is selected may be listed. With regardto the purine analog, a substance which is known per se may be usedwhere 8-azaguanine and decoynine may be exemplified and advantageouslyused. To be more specific, it is reported that, in the case of B.subtilis, said mutant cell can be achieved by preparing a mutant havinga resistance to purine analogs such as 8-azaguanine and decoynine andother antagonists such as methionine sulfoxide (e.g., Ishii and Shiio,Agric. Biol. Chem. 36:1511-1522, 1972; Matsui, et al., Agric. Biol.Chem. 43:1739-1744, 1979).

[0107] As hereunder, there will be mentioned a preferred embodiment forobtaining a mutant cell having a resistance to 8-azaguanine which is notless than the resistance to 8-azaguanine of the wild type cell in thecase of Escherichia coli. Thus, since Escherichia coli JM101 strainwhich is a host cell has a sensitivity to not less than about 100 μg/mlof 8-azaguanine and to about 500 μg/ml of decoynine, Escherichia coliJM101 strain which is subjected to a mutation treatment withN-methyl-N-nitro-nitrosoguanidine using a minimum agar medium containingabout 100 μg/ml of 8-azaguanine or about 500 μg/ml of decoynine isplated whereupon resistant strain for each of them can be obtained.

[0108] It is possible by a genetic engineering manner to prepare a hostcell having a GTP synthesizing ability which is not less than the GTPsynthesizing ability of a wild type cell of the above-mentioned hostcell. Thus, a recombinant cell where the gene for increasing the GTPsynthesizing ability such as the gene of enzyme of the GTP biosynthesissystem is introduced into a wild type cell or a mutant cell such as thathaving a highly GTP-synthesizing ability may be used as a host cell.With regard to the gene which improves the GTP synthesizing ability,that which is known per se may be used. With regard to the said gene,one type may be introduced into a wild type or mutant cell or two ormore genes may be introduced into a wild type or mutant cell. To be morespecific, with regard to a method for the preparation of recombinantcell where the GTP synthesizing ability is improved, there may beexemplified a method where guaBA gene encoding IMP dehydrogenase and GMPsynthase carrying out a conversion of inosinic acid to guanylic acid(Tiedeman, A. A., et al. J. Biol. Chem. 260:8676-8679, 1985 and NucleicAcids Res. 13:1303-1316, 1985 [GenBank M10101]) are introduced into awild type or mutant cell. A method for the introduction of gene such asguaBA into a wild type or mutant cell may be easily carried out by amethod which is known per se such as the following method.

[0109] In the present invention, gene of at least one kind of enzymeparticipating in the THBP synthesis may be introduced into theabove-mentioned host cell. Thus, all genes of the enzymes participatingin the THBP synthesis may be introduced or only gene of a part of thesaid enzymes may be introduced. To be specific, the cases where (a)genes of GCH, PTPS and SPR, (b) two genes from GCH, PTPS and SPR and (c)gene of any of GCH, PTPS and SPR are/is introduced may be exemplified.

[0110] It is also preferred to increase the expressed amount byintroducing the gene of the same enzyme for plural times. It isparticularly preferred that GCH gene is introduced for plural times andorigins of the GCH gene may be same or different.

[0111] A preferred example of the present invention is to introduce thegenes of PTPS and SPR because GCH is present in many host cells such asEscherichia coli and yeast as an enzyme participating in the synthesisof folic acid.

[0112] Another preferred embodiment is that, since the presence of PTPSin addition to GCH is reported, for example, in Synechocystis which is aphotosynthetic Gram-negative bacterium (Lee, S. W. et al., FEMSMicrobiology Letter 176:169-176, 1999), gene of SPR only is introducedinto such a host cell.

[0113] It is also possible that the gene of enzyme participating in theTHBP synthesis is introduced into a cell which inherently has aproducing ability of the biopterin compound. That is because, as aresult, enzymatic activity is enhanced and producing ability of thebiopterin compound is enhanced.

[0114] In the case of production of the biopterin compound where gene oftwo kinds of enzymes—PTPS and SPR—are introduced and expressed while,with regard to GCH, that which is owned by the host cell is used, it ispreferred, as a host cell, to use a mutant cell having an enzymaticactivity of not less than the intrinsic GTP cyclohydrase I of the wildtype cell has.

[0115] With regard to a method for obtaining such mutant cell, there areexemplified a method where mutant of promoter of GCH gene existing inchromosome is obtained, a method where new promoter is introduced intoan upper stream of GCH gene in chromosome and a method where specificactivity is enhanced as compared with that of the wild type cell as aresult of mutation of structural gene of GCH.

[0116] With regard to a method for the introduction of gene of enzymebeing participated in the tetrahydrobiopterin biosynthesis into theabove-mentioned host cell, methods which are known per se may be used.Specific examples of such methods are (a) a method where the said geneis integrated into chromosome of the host cell and (b) a method wheregene is made present as a plasmid using vector. Among them, the methodof using vector is preferred from a viewpoint that DNA can beefficiently introduced.

[0117] With regard to the above-mentioned method (a) according to thepresent invention where the gene of the above-mentioned enzyme isintegrated into chromosome of the host cell, there is exemplified amethod where one end of a glass tube is made narrow by pulling, DNA isplaced thereinto, penetrated into cell and is introduced either byelectrophoretic means or by the pressure caused by sending air ornitrogen gas. Another example is a particle gun method where very fineparticles of gold or silver are sprinkled with DNA and the particlesadhered with DNA are shot to the host cell by means of gun powder orhigh-pressure gas so that DNA is introduced thereinto. Still anotherexample is an electroporation where host cell and DNA are placed in acontainer and voltage is applied whereby a transient pore is resulted inthe host cell and DNA is incorporated therein [Neumann, E., et al. EMBOJ. 1, 841-845 (1982)].

[0118] With regard to a method for the introduction of gene concerningthe present invention into a host cell using vector, a method which isknown per se may be used. With regard to a vector, it is preferred touse expression vector which gives stable mRNA in large quantities and ismade so as to efficiently translate the resulting mRNA in the host cell.When plural genes are introduced, it is desired to use vectors havingdifferent origin for replication both in the case of same genes ordifferent genes. With regard to the vector used, there are exemplifiedplasmid derived from Escherichia coli (such as pBR322, pBR325, pUC12,pUC13, pUC18, pUC19 and pSTV28), plasmid derived from Bacillus subtilis(such as pUB10, pTP5 and pC194), plasmid derived from yeast (such aspSH19, pSH15 and pYES2), bacteriophage such as λ phage, animal virusessuch as retrovirus, vaccinia virus and baculovirus, pA1-11, pXT1,pRc/CMV, pRc/RSV and pcDNAI/Neo.

[0119] Expression vector usually contains regulatory sequence so thatthe enzyme of the present invention is expressed or expressionadvantageously takes place. Each regulatory sequence may be native orforeign to the base sequence coding for the amino acid sequence of theenzyme protein. Such a regulatory sequence includes promoter, leader,polyadenylated sequence, propeptide sequence, enhancer, signal sequence,splicing signal, poly A added signal, SV40 duplicated origin(hereinafter, may be referred to as SV40ori) and transcriptionterminator although not limited thereto. Among the above, the preferredregulatory sequence contains at least promoter and transcription- andtranslation termination signals.

[0120] With regard to promoter, anything may be used so far as it is anappropriate promoter sequence which is a base sequence beingrecognizable by the host cell. When the host cell is bacterium of genusEscherichia, there may be exemplified trp promoter, trc promoter, lacpromoter, recA promoter, λ PL promoter, lpp promoter and T7 promoter.Among those, preferred ones are trc promoter and lac promoter. When thehost cell is a bacterium of genus Bacillus, there may be exemplifiedSPO1 promoter, SPO2 promoter and penP promoter. When the host cell is abacterium of order Actinomycetales, there may be exemplified tipA whichis a promoter inducing an antibiotic thiostrepton (Murakami, T., et al.(1989) J. Bacteriol., 171, 1459), etc.

[0121] When the host cell is yeast, there may be exemplified PHO5promoter, PGK promoter, GAL promoter, GAP promoter, ADH promoter andAOX1 promoter. Among them, GAL promoter is preferred.

[0122] When the host cell is filamentous fungus, there may beexemplified promoter obtained from gene coding for TAKA amylase ofAspergillus oryzae, aspartic acid proteinase of Rhizomucor miehei,neutral a-amylase of Aspergillus niger, acid-stable α-amylase ofAspergillus niger, glucoamylase (glaA) of Aspergillus niger orAspergillus awamori, lipase of Rhizomucor miehei, alkaline protease ofAspergillus oryzae, triphosphoric acid isomerase of Aspergillus oryzae,acetamidase of Aspergillus nidulans and trypsin-like protease ofFusarium oxysporum (U.S. Pat. No. 4,288,627 and Japanese PatentLaid-Open No. 507102/2000).

[0123] When the host cell is animal cell, there may be exemplified SRαprompter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoterand HSV-TK promoter.

[0124] When the host cell is insect cell, there may be exemplifiedpolyhedrin promoter and P10 promoter.

[0125] When the host cell is plant cell, there may be exemplified 35Spromoter of cauliflower mosaic virus.

[0126] With regard to transcription terminator, any sequence may be usedso far as it is recognized by a host cell for terminating thetranscription.

[0127] For example, when the animal cell is a host, there may be usedtranscription terminator sequence of each of genes derived from virus,various mammals and birds and, to be more specific, there may be usedSV40 terminator of simian virus.

[0128] When the host cell is yeast, there may be exemplified, PHO5terminator, PGK terminator, GAL terminator, GAP terminator, ADHterminator and AOX1 terminator.

[0129] When the host cell is filamentous fungus, there may beexemplified terminators obtained from the gene coding for TAKA amylaseof Aspergillus oryzae, glucoamylase of Aspergillus niger, anthranilatesynthase of Aspergillus nidulans, α-glucosidase of Aspergillus niger andtrypsin-like protease of Fusarium oxysporum.

[0130] Expression vector may include signal sequence concerningsecretion of protein.

[0131] With regard to signal sequence, a signal sequence of the gene tobe introduced may be used or a signal sequence of the different gene maybe used.

[0132] With regard to the signal sequence of the different gene, theremay be exemplified alkaline phosphatase when the host cell is abacterium of genus Escherichia and, when the host cell is a bacterium ofgenus Bacillus, there may be exemplified a-amylase signal sequence andsubtilisin signal sequence.

[0133] When the host cell is yeast, there may be exemplified MFα signalsequence and SUC2 signal sequence.

[0134] When the host cell is filamentous fungus, there may beexemplified base sequences coding for signal peptide from Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene,Rhizomucor miehei aspartic acid proteinase gene, Humicola lanuginosacellulose gene and Rhizomucor miehei lipase gene.

[0135] When the host cell is animal cell, there may be exemplifiedinsulin signal sequence, a-interferon signal sequence and antibodymolecule signal sequence.

[0136] Expression vector may include a selective marker. For example, inthe case of prokaryote such as Escherichia coli and Bacillus subtilis,there may be used various drug-resistant genes while, in the case ofeukaryotic microbe such as yeast, there may be used gene complementaryto auxotrophy of the host as a selective marker. To be more specific,there may be exemplified dihydrofolic acid reductase gene, methotrexate(MTX)-resistant gene, ampicillin-resistant gene, neomycin-resistant gene(G418-resistant), chloramphenicol-resistant gene, kanamycin-resistantgene and URA3 gene.

[0137] Expression vector may include one or more nucleic acidsequence(s) coding for one or more factor(s) (such as activator (forexample, trans-acting factor), chaperone, SD sequence and processingprotease) which is/are advantageous for expression of enzyme geneconcerning the present invention.

[0138] Any factor which is functional in the selected host cell may beused as an expression vector concerning the present invention.

[0139] In the present invention, known expression vector may be used aswell. For example, there may be listed PIN-III-ompA₂, etc. forEscherichia coli host cell. Further examples are pIJ702 for Actinomyceshost cell and pNJ1053 for yeast host cell. When plant cell is host cell,there may be exemplified pBI121 (Nucleic Acids Res., 12, 8771-8721(1984)), etc. In addition, shuttle vector pDG148 (Karmazyn-Campelli, etal., Cell, 52, 697-704, 1988) may be used for Escherichia coli andBacillus subtilis and are advantageously used in the present invention.

[0140] With regard to a method for integrating cDNA, etc. containing thegene of enzyme participating in the above THBP biosynthesis concerningthe present invention, there may be used a method which is known per sesuch as cleaving by a restriction enzyme followed by bonding by a DNAligase. With regard to the above-mentioned cDNA, etc., varioustreatments may be previously carried out such as treatment withexonuclease, addition of chemically synthesized DNA fragment andaddition of cleavage sites for restriction enzymes by bonding of linkeror by PCR so as to be easily integrated with the above vector orG,C-chain is elongated so as to add a connectable terminus at theterminus of vector DNA or double stranded cDNA.

[0141] When the vector containing the gene of enzyme which is able toparticipate in the THBP biosynthesis constructed as such is introducedinto a host cell, it is possible to manufacture a transformed cell inaccordance with the present invention.

[0142] With regard to a method for the transformation by introduction ofthe said expression vector into the host cell, known methods may beused. When the host cell is prokaryote such as bacterium of genusEscherichia, the introduction may be carried out by recovering the cellsin a logarithmic growth phase followed by subjecting to a well-knownCaCl₂ method (Graham, F. L. and van der Eb, A. J. Virology, 52, 456-467(1973)). When MgCl₂ or RbCl is made coexistent in the transformationreaction solution, the transforming efficiency can be improved and,therefore, that may be coexisted in the present invention. It is alsopossible to carry out a transformation after preparation of protoplastof the host cell.

[0143] When the host cell used is eukaryote, it is possible to carry outthe introduction by common methods such as a method where DNA isinfected as a calcium phosphate precipitate, a microinjection method, amethod where introduction is carried out by including plasmid witherythrocyte cell or liposome, a method of treating the cell with areagent such as lysophosphatidylcholine and a method where virus vectoris used. When the host cell is yeast, it is possible to use a lithiumacetate method.

[0144] To be more specific, in the transformation of bacterium of genusEscherichia, that may be carried out by the methods described in Proc.Natl. Acad. Sci. USA, volume 69, 2110 (1972), Gene, volume 17, 107(1982), etc.

[0145] In the transformation of bacterium of genus Bacillus, that may becarried out by the methods described in Molecular & General Genetics,volume 168, 11 (1979), etc.

[0146] In the transformation of yeast, that may be carried out by themethods described in Methods in Enzymology, volume 194, 182-187 (1991),Proc. Natl. Acad. Sci. USA, volume 75, 1929 (1978), etc.

[0147] In the transformation of insect cell or insect, that may becarried out by the methods described in Bio/Technology, 6, 47-55 (1988),etc.

[0148] In the transformation of animal cell, that may be carried out bythe methods described in Saibo Kogaku, Supplementary Issue No. 8, ShinSaibo Kogaku Jikken Protocol, 263-267 (1995) (published by Shujunsha),Virology, volume 52,456(1973), etc.

[0149] In the transformation of plant cell, that may be carried out byintroducing the gene in accordance with the present invention accordingto an Agrobacterium tumefaciens method (Methods in Enzymol., 118,627-640 (1986)), a high-speed fine particle method (Plant MolecularBiology, 11, 433-439 (1989)), a protoplast method (Nature, 319, 791-793(1986)), etc.

[0150] With regard to a method for a stable expression of the enzyme ofthe present invention using animal cell, there is a method where thecell in which the expression vector introduced into the above animalcell is integrated with chromosome is selected by means of a cloneselection. To be more specific, transformed cell is selected using theabove-mentioned selecting marker as an index and the transformed cellwhich is obtained by the selective marker as such is repeatedlysubjected to a clone selection whereupon a stable transformed cellhaving a high enzyme expressing ability according to the presentinvention is able to be obtained.

[0151] In the present invention, the above-mentioned transformed cell iscultured usually under such a condition that gene of enzymesparticipating in the THBP synthesis such as GCH, PTPS and SPR introducedtherein is able to be expressed. In the cultivation, it is preferredthat incubating temperature, pH of medium and dissolved oxygen level areconstantly controlled. That is because, in order to suppress a reductionof growth of cell by, for example, lowering of pH of the medium as muchas possible, to promote the growth and also to produce the biopterincompound more efficiently, it is preferred to make the cultureconditions constant as mentioned above.

[0152] In the culture of transformed cell where the host cell is abacterium of genus Escherichia, a bacterium of genus Bacillus or abacterium of order Actinomycetales, a liquid medium is preferred as amedium used for the culture and the medium in which carbon source,nitrogen source, inorganic material and others necessary for the growthof the said transformed cell are contained is preferred. Examples of thecarbon source are glucose, dextrin, soluble starch and sucrose; examplesof the nitrogen source are ammonium salt, nitrate, corn steep liquor,peptone, casein, meat extract, soybean cake and potato extract and otherinorganic or organic substances; and examples of the inorganic materialare calcium chloride, sodium dihydrogen phosphate and magnesiumchloride. Yeast extract, vitamins, growth-promoting factor, etc. mayalso be added to the above medium. PH of the medium is preferably to beabout 5 to 8.

[0153] With regard to the medium for the culture of a bacterium of genusEscherichia, its specific examples are M9 medium containing glucose andCasamino acid (Miller, Journal of Experiments in Molecular Genetics,431-433, Cold Spring Harbor Laboratory, New York, 1972), etc. and it ispreferred that, after a pre-culture using an LB medium (refer to Example4), a main culture is carried out using an NUCA medium (refer to Example4). Here, in order to have a promoter act efficiently, a reagent such as3β-indolylacrylic acid or chloramphenicol may be added thereto ifnecessary. When an inductive promoter is used, it is preferred to add asubstance which causes the induction to the medium. For example, in thecase of lac promoter, it is preferred to add isopropylβ-thiogalactopyranoside (IPTG) and, in the case of GAL promoter, it ispreferred to add galactose. The culture is carried out preferably atabout 10 to 50° C. for about 3 to 72 hours and, if desired, aeration orstirring may be conducted.

[0154] Culture of a bacterium of genus Bacillus is usually carried outat about 30 to 40° C. for about 6 to 40 hours and, if desired, aerationand stirring may be conducted. With regard to the medium, known ones maybe used. To be more specific, it is preferred that, for example, apre-culture is carried out using an LB medium (refer to Example 4) andthen a main culture is carried out using an NU medium (refer to Example14).

[0155] When the host cell is a bacterium of order Actinomycetales, it isusually carried out at about 20 to 40° C. for about 2 to 7 days and, ifdesired, aeration or stirring may be conducted. With regard to themedium, it is possible to use known media such as a GP medium(containing 0.4 wt % of glycerol, 0.1 wt % of peptone, 0.4 wt % of yeastextract, 0.05 wt % of magnesium sulfate, 0.2 wt % of monopotassiumphosphate, 0.5 wt % of disodium phosphate and 0.1 wt % of glycine perone liter).

[0156] With regard to the medium for the culture of yeast, there may beexemplified a Burkholder minimum medium [Bostian, K. L., et al. Proc.Natl. Acad. Sci. USA, volume 77, 4505 (1980)] and an SD mediumcontaining 0.5% of Casamino acid [Bitter, G. A., et al., Proc. Natl.Acad. Sci. USA, volume 81, 5330 (1984)]. Among those, an SD-Ura⁻ medium(refer to Example 5) is preferred. It is preferred that pH of the mediumis adjusted to about 5-8. Culture is preferably carried out at about20-40° C. for about 24-84 hours and, if desired, aeration or stirringmay be conducted.

[0157] When the host cell is a filamentous fungus, it is also possibleto culture by a method known per se.

[0158] When a transformed cell where the host cell is insect cell orinsect is cultured, examples of the medium are that where an additivesuch as inactivated 10% bovine serum is appropriately added to Grace'sinsect medium (Grace, T. C. C., Nature, 195, 788 (1962)), etc. It ispreferred that pH of the medium is adjusted to about 6.2 to 6.4. Cultureis preferably carried out at about 27° C. for about 3 to 5 days and, ifdesired, aeration or stirring may be conducted.

[0159] With regard to a medium for the culture of animal cell, there maybe used an MEM medium containing about 5 to 20% of fetal bovine serum(Science, volume 122, 501 (1952)), a DMEM medium (Virology, volume 8,396 (1959)), an RPMI 1640 medium (J. Amer. Med. Assc., volume 199, 519(1967)), a 199 medium (Proc. Soc. Biol. Med., volume 73, 1 (1950)), etc.It is preferred that pH of the medium is about 6 to 8. Culture ispreferably carried out at about 30 to 40° C. for about 15 to 72 hours.

[0160] Examples of the medium for the culture of plant cell are aMurashige and Skoog (MS) medium, a White medium, etc.

[0161] As hereunder, preferred embodiments of the manufacturing methodfor biopterin compound according to the present invention will bementioned.

[0162] The pSTV28 is used as a vector of an Escherichia coli type andthere is prepared pSTV28-GPS which is a plasmid where cDNA of each ofGCH, PTPS and SPR is aligned at the downstream of a lactose promoter.The said plasmid is an expression vector where an induction by IPTG ispossible. The expression vector is introduced into an Escherichia coliJM 101 strain and cultured in a medium containing 0.5 mM IPTG for about48 hours whereby the biopterin compound can be manufactured.

[0163] Alternatively, yeast pYES2 is used as a vector and there isprepared pYES2-FPS which is a plasmid where cDNA of each of GCH, PTPSand SPR is aligned at the downstream of a GAL1 promoter. Such a plasmidis an expression vector where an expression induction by galactose ispossible. The expression vector is introduced into Saccharomyces yeastand expression of each enzyme gene is carried out by induction withgalactose whereby the biopterin compound can be manufactured.

[0164] There is also the following embodiment as a method for themanufacture of the biopterin compound according to the presentinvention. Thus, a shuttle vector pDG148 (Karmazyn-Campelli, et al.,Cell, 52, 697-704, 1988) is used and there is prepared pDG148 MPS whichis a plasmid where cDNA of each of GCH, PTPS and SPR is aligned at thedownstream of spac promoter. The said plasmid is an expression plasmidfor Bacillus subtilis which is able to be induced by IPTG. Theexpression vector is introduced into Bacillus subtilis 1A1 strain(trpC2) to prepare a transformed cell. Such a transformed cell ispre-culture in an LB medium containing about 5 μg/ml of kanamycin forabout 3 hours and then subjected to a shaking culture in an NU mediumcontaining about 1 mM of IPTG and about 5 μg/ml of kanamycin at about37° C. for about 20 hours whereby the biopterin compound can bemanufactured.

[0165] It is also possible that, when lacI gene is deleted from theabove-mentioned shuttle vector pDG148, an expression vector pDG148 MPSAIwhich is able to always express GCH, PTPS and SPR regardless of presenceor absence of IPTG is prepared. Such an expression vector may be used bythe same manner as above except that IPTG is not necessary during themain culture.

[0166] THBP is produced from GTP which is inherently present in hostcell by the enzyme which is able to participate in the THBP synthesissuch as GCH, PTPS or SPR expressed in the transformed cell. The producedTHBP is oxidized to DHBP as shown in FIG. 2 and then further oxidized toBP in the cell or after discharged outside the cell by passing throughcell membrane in a medium such as a culture broth (Takikawa, et al.,Eur. J. Biochem. 161:295-302, 1986). Thus, although an indiscriminateconclusion cannot be derived because of the difference due to type ofthe transformed cell and extracellular environment such as culturecondition, only THBP is produced in the transformed cell in some caseswhile, in other cases, a part of or all of it is oxidized and DHBP or BPor a mixture thereof is available. There is also another case where theTHBP produced in the transformed cell passes through a cell membrane andis discharged outside the cell. In that case, although an indiscriminateconclusion cannot be derived because of the difference due tocomposition of the culture media, the discharged THBP is sometimesoxidized to DHBP or BP in the culture media.

[0167] In the present invention, the biopterin compound in the culturebroth or in the treated substance thereof such as a supernatant may bepurified and separated by the following method. If desired, the culturebroth or the treated substance thereof such as a supernatant is oxidizedby, for example, adding an oxidizing agent thereto and, after that, thebiopterin compound is purified and separated. Since THBP is an easilyoxidized substance among the biopterin compounds, it is preferred thatthe culture broth or the treated substance thereof such as a supernatantis oxidized so that THBP or DHBP is oxidized to BP and, after that,chemically more stable BP is purified and separated.

[0168] With regard to a method for the oxidation of the biopterincompound in the culture broth or in the treated substance thereof suchas a supernatant, known method per se may be used and a known oxidizingagent may be added to the culture broth or to the treated substancethereof such as a supernatant. Examples of the oxidizing agent areperiodate such as potassium iodide, potassium or sodium dichromate,potassium permanganate, potassium nitrosodisulfonate and nitric acidand, among them, it is preferred to use potassium iodide.

[0169] In order to separate and purify the biopterin compound of thepresent invention from the above culture product, methods which areknown per se may be used.

[0170] To be more specific, there may be appropriately used, forexample, a method where fungus body or cell is collected by a knownmethod after culture in extraction of the biopterin compound of thepresent invention from cultured fungus body or cell which is transformedcell and is suspended in an appropriate buffer followed by subjecting toa treatment with ultrasonic wave or lysozyme or to a freeze-melting anda method where fungus body or cell is destroyed by a combination of sucha means, then transformed cell and supernatant of culture broth areseparated by a means which is known per se such as centrifugation orfiltration and the supernatant is collected wherefrom a solution inwhich the biopterin compound of the present invention is dissolved isobtained.

[0171] When THBP produced in the transformed cell is discharged outsidethe cell by passing through a cell membrane, there may be appropriatelyused, for example, a method where transformed cell and supernatant areseparated by a known means per se such as centrifugation or filtrationwithout destroying the transformed cell and the supernatant is collectedwherefrom the solution in which the biopterin compound of the presentinvention is dissolved is obtained.

[0172] Purification of the biopterin compound of the present inventionwhich is contained in the culture broth or the treated product thereofsuch as supernatant prepared as such may be carried out by anappropriate combination of separating and purifying methods which areknown per se.

[0173] With regard to such known methods for separation andpurification, there may be used a method where solubility is utilizedsuch as salting out and solvent precipitation; a method where differencein molecular weights is mainly utilized such as dialysis,ultrafiltration, gel filtration and SDS-polyacrylamide gelelectrophoresis; a method where difference in charges is utilized suchas ion-exchange chromatography; a method where specific affinity isutilized such as affinity chromatography; a method where difference inhydrophobicity is utilized such as a reversed phase high performanceliquid chromatography; a method where difference in isoelectric point isutilized such as isoelectric focusing; and the like.

[0174] When the biopterin compound of the present invention obtained assuch is obtained in a free form, it may be converted to a salt by aknown method per se or by a method similar to that and, conversely, whenit is obtained in a form of salt, it may be converted to a freesubstance or to other salt by a known method per se or by a methodsimilar to that.

[0175] Preferred embodiment of purification of the biopterin compound ofthe present invention is a method where the culture broth is oxidizedwith a potassium iodide solution under an acidic condition to convert toBP and then pure BP is obtained by way of precipitating operation, Dowex1×8 chromatography and Florisil chromatography.

[0176] The biopterin compound which is obtained as such is able to beconverted to THBP, if desired, using a known means. For example, BP orDHBP can be converted to THBP by a chemical hydrogenation reaction. Inthe chemical hydrogenation reaction, a known method per se may be usedand there may be exemplified a method where the reaction is carried outwith lithium aluminum hydride, lithium boron triethyl hydride, sodiumboron hydride, diborane, alkyl diborane, etc. and a method wherereduction is carried out using Raney nickel catalyst. Reaction conditiontherefor may be in accordance with the known methods. Under somereduction conditions, it is also possible to obtain DHBP from BP.

[0177] As mentioned above, THBP has been known as a coenzyme for variouskinds of enzymes and is a substance expected to be a substance havingsuch a pharmacological action. In addition, DHBP or BP is not onlyuseful as a provider for such a THBP but also is a useful substancehaving a possibility of exhibiting a pharmacological action.

Examples

[0178] Examples of the present invention will be shown as follows.Incidentally, the following basic operations of genetic engineering orbiological engineering were carried out according to the methodsmentioned in “Molecular Cloning” (Cold Spring Harbor Laboratory, 1982);“Molecular Cloning” 2nd edition (Cold Spring Harbor Laboratory, 1989);Methods in Enzymol., volume 194 (1991); Jikken Igaku (SupplementaryIssue), Kobo ni yoru Idenshi Jikkenho (Methods for Genetic ExperimentsUsing Enzymes), Yodosha (1994); etc. When a commercially available kitwas used, the instructions attached thereto were followed.

Example 1 Preparation of GCH Gene, PTPS Gene and SPR Gene

[0179] 1. Cloning of GCH (GTP cyclohydrase I) gene derived fromEscherichia coli

[0180] Genomic DNA was extracted from Escherichia coli (W3110 strain) bya reported method (Seibutsu Kogaku Jikkensho [published by Baifukan,pages 97-98]). This was used as a template and a PCR was carried out bya conventional manner using sense primer P1 (SEQ ID NO: 1) and antisenseprimer P2 (SEQ ID NO: 2) to prepare GCH gene (folE) [Katzenmeier, G., etal., Bio Chem Hoppe Seyler 372:991-997, 1991, [GenBank X63910]]. Afterthat, DNA containing the resulting GCH gene was used as a template and aPCR was carried out using sense primer P3 (SEQ ID NO: 3) and antisenseprimer P4 (SEQ ID NO: 4) to add cleavage sites for restriction enzymesEcoRI and SpeI to the untranslated regions of 5′end and 3′end of GCHgene, respectively. This PCR product was digested by EcoRI and SpeI andintroduced into the EcoRI and SpeI sites of vector of pProEX HTa (GIBCOBRL) to prepare pProEX-GCH. Incidentally, PCR condition, treatment withrestriction enzyme and ligation reaction were carried out according toconventional methods.

[0181] After that, a PCR was carried out using pProEX-GCH as a templateand using sense primer P5 (SEQ ID NO: 5) and antisense primer P6 (SEQ IDNO: 6) for cloning the Escherichia coli GCH gene which was cloned topProEX-GCH to a plasmid pSTV28 (Takara Shuzo) to prepare GCH gene havingcleavage sites for the restriction enzymes EcoRI and SailI at thetermini of 5′end and 3′end, respectively. The resulting PCR product wascleaved by restriction enzymes EcoRI and SailI and connected to EcoRIand SalI fragments (3.0 kb) of pSTV28 (hereinafter, may be sometimesreferred to as EcoRI-SalI fragment or EcoRI, SalI fragment; that will beapplied to others as well) to give Escherichia coli GCH expressionplasmid pSTV28-GCH (FIG. 3). The GCH gene (folE) contained in pSTV28-GCHwas transcribed by Escherichia coli lactose (lac) promoter and the GCHexpressed hereby had an amino acid sequence where 7 amino acids(underlined part in FIG. 10) derived from pSTV28 were added to the aminoterminus (FIG. 10).

[0182] 2. Cloning of PTPS (6-pyruvoyltetrahydropterin Synthase) GeneDerived from Rat

[0183] Liver excised from rat was treated with collagenase and theresulting hepatic cell was treated with a TRIzol reagent (GIBCO BRL) toextract total RNA. The total RNA from rat hepatocyte treated with DNasewas subjected to an RT-PCR using Super Script Preamplification System(GIBCO BRL) to prepare cDNA containing PTPS gene (Inoue, Y., et al., J.Biol. Chem. 266:20791-20796, 1991: [GenBank NM_(—)017220]). Here, oligodT primer was used for reverse transcription while, for amplification ofa single stranded DNA containing PTPS gene by PCR, there were used senseprimer P7 (SEQ ID NO: 7) and antisense primer P8 (SEQ ID NO: 8). A PCRwas carried out using the resulting cDNA containing PTPS as a templateand using sense primer P9 (SEQ ID NO: 9) and antisense primer P10 (SEQID NO: 10) to add the cleavage sites for restriction enzymes EcoRI andSpeI to the untranslated regions at 5′end and 3′end, respectively, ofcDNA containing PTPS gene. The PCR product was digested by EcoRI andSpeI to insert into the EcoRI and SpeI sites of pProEX HTc vector (GIBCOBRL) whereupon pProEX-PTPS was prepared (FIG. 4).

[0184] After that, a PCR was carried out using pProEX-PTPS as a templateand using sense primer P11 (SEQ ID NO: 11) and antisense primer P12 (SEQID NO: 12) to give PTPS gene having the cleavage sites for therestriction enzymes EcoRI and SalI. The resulting PCR product wascleaved by the restriction enzymes EcoRI and SalI and connected to EcoRIand SalI fragment (2.7 kb) of pUC18 (Yanisch-Perron, C., Vieira, J. andMessing, J. Gene, 33:103-119, 1985) to give rat PTPS expression plasmidpUC18-PTPS (FIG. 4). The PTPS gene contained in pUC18-PTPS wastranscribed by Escherichia coli lac promoter and the expressed PTPS hadan amino acid sequence where 7 amino acids (underlined ones in FIG. 11)derived from pUC18 were added to amino terminus (FIG. 11).

[0185] 3. Cloning of SPR (Sepiapterin Reductase) Gene Derived from Rat

[0186] According to the same method as in the case of cDNA containingPTPS gene, there was prepared cDNA containing SPR gene (Citron, B. A.,et al. Proc. Natl. Acad. Sci. USA 87:6436-6440, 1990, [GenBank M36410]with an exception that sense primer P13 (SEQ ID NO: 13) and antisenseprimer P14 (SEQ ID NO: 14) were used for amplification of singlestranded DNA containing SPR gene by means of a PCR. The cDNA containingSPR gene was used as a template and a PCR was carried out using senseprimer P15 (SEQ ID NO: 15) and antisense primer P16 (SEQ ID NO: 16) toadd the cleavage sites for the restriction enzymes BamHI and SpeI to theuntranslated regions of 5′end and 3′end, respectively, of cDNAcontaining SPR gene. The PCR product was digested by BamHI and SpeI andinserted into the BamHI and SpeI sites of pProEX HTb vector (GIBCO BRL)to prepare pProEX-SPR (FIG. 5).

[0187] After that, a PCR was carried out for rat SPR gene usingpProEX-SPR as a template and using sense primer P17 (SEQ ID NO: 17) andantisense primer P18 (SEQ ID NO: 18) to prepare SPR gene having thecleavage sites for a restriction enzyme HindIII at termini of 5′end and3′end, respectively and the resulting PCR product was cleaved by arestriction enzyme HindIII and connected to HindIII fragment (2.7 kb) ofpUC19 (Yanisch-Perron, C., Vieira, J. and Messing, J. Gene, 33:103-119,1985) to prepare a rat SPR expression plasmid pUC19-SPR (FIG. 5). Withregard to SPR gene coded to pUC19-SPR, it was also transcribed byEscherichia coli lac promoter and the expressed SPR had an amino acidsequence where 8 amino acids (the underlined one in FIG. 12) derivedfrom pUC19 were added to amino terminus (FIG. 12).

[0188] 4. Cloning of GCH Gene (FOL 2) Derived from Yeast

[0189] FOL2 is a homolog of GCH. A PCR was carried out using genomic DNAof yeast (Saccharomyces cerevisiae, KA31 strain) as a template and usingsense primer P23 (SEQ ID NO: 23) and antisense primer P24 (SEQ ID NO:24) to give DNA containing FOL2 gene (Tettelin, H., et al. Nature387:81-84, 1997, [GenBank NC_(—)001139]) having the cleavage sites forrestriction enzymes BamHI and XhoI of the untranslated region at the5′end and 3′end, respectively. The PCR product was digested by BamHI andXhoI and inserted into the BamHI and XhoI sites of pYES2/CT vector(Invitrogen) to prepare pYES2-FOL2 (FIG. 6).

Example 2 Preparation of Plasmid pSTV28-GPS Producing the BiopterinCompound for Escherichia coli

[0190] pSTV28-GPS which is a THBP synthase expression plasmid forEscherichia coli was prepared by the following method. Firstly, a PCRwas carried out using pUC18-PTPS mentioned in Example 1 as a template toamplify DNA containing from lac promoter to termination codon of PTPSgene. In designing the primer for the PCR, SalI site was provided tosense primer while BamHI site was provided to antisense primer so as tomake the cloning thereafter easy. Those primers had sequences of senseprimer P19 (SEQ ID NO: 19) and antisense primer P20 (SEQ ID NO: 20),respectively. The resulting PCR product was subjected to a precipitatingtreatment with ethanol, dissolved in a TE buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA) and cleaved by restriction enzymes SalI and BamHI.

[0191] After that, a PCR was carried out using pUC19-SPR mentioned inExample 1 as a template to amplify the DNA containing from lac promoterto termination codon of SPR gene. In the primers for the PCR, BamHI sitewas provided to sense primer while SphI site was provided to antisenseprimer. Those primers had a sequence of sense primer P21 (SEQ ID NO: 21)and that of antisense primer P22 (SEQ ID NO: 22), respectively. Theresulting PCR product was subjected to a precipitation treatment withethanol, dissolved in a TE buffer (10 mM Tris-HCl (pH 8.0) and 1 mMEDTA) and cleaved by restriction enzymes BamHI and SphI.

[0192] The SalI-BamHI fragment containing PTPS gene and BamHI-SphIfragment containing SPR gene prepared as such were connected to digestedproduct (3.9 kb) of pSTV28-GCH by XhoI and SphI to prepare pSTV28GPS(FIG. 7). The pSTV28-GPS has a structure where each of GCH, PTPS and SPRgenes (described in FIG. 10 to FIG. 12) is transcribed by lac promoter.

Example 3 Preparation of pYES2-FPS, a Plasmid Producing the BiopterinCompound for Yeast

[0193] A PCR was carried out using sense primer P25 (SEQ ID NO: 25) andantisense primer P26 (SEQ ID NO: 26) where pUC18-PTPS was used as atemplate to add the cleavage sites for restriction enzymes BamHI andXhoI to the untranslated region at 5′end and 3′end, respectively, ofPTPS gene. The PCR product was digested by BamHI and XhoI and insertedinto BamHI and XhoI sites of pYES2/CT vector to prepare pYES2-PTPS (FIG.8).

[0194] After that, a PCR was carried out using sense primer P27 (SEQ IDNO: 27) and antisense primer P28 (SEQ ID NO: 28) where pUC19-SPR wasused as a template to prepare cDNA containing SPR gene having thecleavage sites for restriction enzymes BamHI and XhoI at theuntranslated region at 5′end and 3′end, respectively, of SPR gene. ThePCR product was digested by BamHI and XhoI and inserted into the BamHIand XhoI sites of pYES2/CT vector (Invitrogen) to prepare pYES2-SPR(FIG. 8).

[0195] Then a PCR was carried out using sense primer P29 (SEQ ID NO: 29)and antisense primer P30 (SEQ ID NO: 30) where pYES2-PTPS was used as atemplate to prepare DNA fragment having GAL1 promoter to which thecleavage site for SphI was added at upper stream of PTPS gene and CYC1transcription termination signal to which the cleavage site for SpeI wasadded at downstream. Incidentally, this DNA fragment has blunt endsbecause of the property of DNA polymerase (pyrobest DNA Polymerase[Takara Shuzo]) used therefor. The said DNA fragment where the5′terminus was phosphorylated was inserted into pYES2-SPR where its endswere blunted after being digested by SpeI to prepare pYES2-PS. Afterthat, a PCR was carried out using sense primer P31 (SEQ ID NO: 31) andantisense primer P32 (SEQ ID NO: 32) where pYES2-FOL2 was used as atemplate to prepare DNA fragment having GAL1 promoter to which thecleavage site for SpeI was added at upper stream of FOL2 gene and CYCItranscription termination signal to which the cleavage site for SpeI wasadded at downstream. The fragment was digested by SpeI and inserted intothe cleavage site for SpeI of pYES2-PS to prepare pYES2-FPS. In thepYES2-FPS, it had a structure where each of GCH gene derived from S.cerevisiae, PTPS gene derived from rat and SPR gene derived from rat istranscribed by GAL1 promoter and enzyme having the same amino acidsequence as the enzyme of a natural type is expressed (FIG. 9).

Example 4 Production of the Biopterin Compound by Escherichia coli

[0196]Escherichia coli JM101 strain was transformed using pSTV28-GPS bya calcium chloride method (Mandel and Higa, J. Mol. Biol., 53, 159-162,1970) and the resulting JM101/pSTV28-GPS was cultured to investigate itsability of production of the biopterin compound. Firstly,JM101/pSTV28-GPS cultured for one night in an LB medium was used as apre-culture broth, 50 μl thereof were inoculated in 3 mL of an NUCAmedium containing 0.5 mM IPTG (isopropyl thiogalactopyranoside) andcultured at 37° C. for 48 hours. Incidentally, the LB medium and NUCAmedium comprised the following compositions. Thus, [composition in 1 Lof the LB medium: 10 g of tryptone, 5 g of yeast extract and 5 g ofNaCl] and [composition in 1 L of NUCA medium: 20 g of glycerol, 4 g ofyeast extract, 10 g of Casamino acid, 4 g of K₂HPO₄, 4 g of KH₂PO₄, 2.7g of Na₂HPO₄, 1.2 g of (NH₄)₂SO₄, 0.2 g of NH₄Cl, 2 g of MgSO₄₀.7H₂O, 40mg of FeSO₄₀.7H₂O, 40 mg of CaCl₂₀.2H₂O, 10 mg of MnSO₄.nH₂O, 10 mg ofAlCl₃₀.6H₂O, 4 mg of COCl₂₀.6H₂O, 2 mg of ZnSO₄₀.7H₂O, 2 mg ofNa₂MoO₄₀.2H₂O, 1 mg of CuCl₂₀.7H₂O, 0.5 mg of H₃BO₄ and 30 mg ofchloramphenicol]

[0197] The resulting THBP is easily oxidized in its aqueous solution togive DHBP and BP and, in order to check whether JM101/pSTV28-GPSproduced the THBP, the culture broth was subjected to an iodineoxidization to convert to BP and the produced amount thereby wasmeasured. The culture broth was centrifuged to remove the cells,one-tenth by volume of potassium iodide solution (1N hydrochloric acidsolution containing 0.9% of 12 and 1.8% of KI) was added to the culturebroth and the mixture was allowed to stand for 1 hour shielding thelight. Then this culture broth was diluted to 20-fold with deionizedwater, injected to a C18 reversed phase column (COSMOSIL 5C18-AR,Nakarai Tesk) of 4.6 mm×250 mm equilibrated with 10 mM sodium phosphatebuffer at a flow rate of 0.8 mL/minute and BP was quantified by excitingat 350 nm using a 440 nm fluorescent detector. As a result, peakscorresponding to standard BP specimen and standard P (pteridine)specimen were detected in the supernatant (FIG. 13) whereby it wasapparent that JM101/pSTV28-GPS produced the biopterin compound. When theamount of the biopterin compound was calculated from the peak areas, theproduction amount per liter of the culture broth was about 20 mg.

Example 5 Production of Biopterin Compound by Yeast

[0198]S. cerevisiae KA31 strain (MATaura3 leu2 his3 trpl) wastransformed by a lithium acetate method using pYES2-FPS. The transformedcell was selected by an SD-Ura⁻ medium to give an FPS strain.[Composition in 1 liter of the SD-Ura medium: 20 g of glucose, 1.7 g ofyeast nitrogen base (containing neither amino acid nor ammoniumsulfate), 5 g of ammonium sulfate, 20 mg of adenine sulfate, 20 mg ofArg, 100 mg of Asp, 100 mg of Glu, 30 mg of Ile, 30 mg of Lys, 20 mg ofMet, 50 mg of Phe, 400 mg of Ser, 200 mg of Thr, 30 mg of Tyr, 150 mg ofVal, 20 mg of His, 100 mg of Leu and 20 mg of Trp]

[0199] Similarly was prepared a transformed cell by vector (pYES2/CT) asa control (Mock).

[0200] The resulting transformed cell was cultured in 5 ml of SCD-Uramedium until OD_(600 nm) became 0.4 and, after that, it was substitutedto the same amount of an SCGal-Ura medium to conduct expressioninduction. Incidentally, the incubating temperature was made 30° C.

[0201] Composition in 1 liter of the SCD-Ura⁻ medium: 1.7 g of yeastnitrogen base (containing neither amino acid nor ammonium sulfate), 5 gof ammonium sulfate, 5 g of Casamino acid, 20 g of glucose, 20 mg ofadenine sulfate and 20 mg of Trp.

[0202] Composition in 1 liter of the SCGal-Ura⁻ medium: 1.7 g of yeastnitrogen base (containing neither amino acid nor ammonium sulfate), 5 gof ammonium sulfate, 5 g of Casamino acid, 20 g of galactose, 20 mg ofadenine sulfate and 20 mg of Trp.

[0203] After 24 hours from the expression induction, yellow colorationwhich was specific to the pteridine compound was noted in the culturebroth of the FPS strain whereby it was noted from the appearance thatthe expressed three kinds of enzymes functioned in the cells.

[0204] Growth of the cells after the induction was good in both FPSstrain and Mock strain (FIG. 14). In order to confirm the production ofTHBP or its oxidized product, the culture supernatant after 72 hoursfrom the induction was oxidized as same as in Example 4 and subjected toHPLC analysis and TLC analysis.

[0205] Incidentally, the HPLC analysis was carried out by the samemethod as in Example 4. In the TLC analysis, a thin layer plate ofsilica gel for thin layer chromatography (silica gel 60 F₂₅₄; layerthickness: 0.25 mm; 10 cm×20 cm) was used and, as to a developingsolvent, a mixture of chloroform, methanol, acetic acid and water(45:10:5:2) was used. The sample (20 μl) and each 100 ng of standard BPand P specimens were spotted onto the thin layer plate, dried with airand developed by an inclined ascending method. When the developeddistance became about 12 cm, development was finished, the thin layerplate was dried with air and irradiated with ultraviolet ray (mainwavelength: 254 nm) and the absorption spot was detected.

[0206] Result of the HPLC analysis is shown in FIG. 15. Only in the FPSstrain, the peak corresponding to BP was noted and, from the area ratioof peak areas, it was found that the FPS strain produced the biopterincompound corresponding to 0.76 μg/ml of BP by induction for 72 hours inthe culture supernatant.

[0207]FIG. 16 shows the result of the TLC analysis of the culturesupernatant. In the culture supernatant of the FPS strain, a spotshowing the same mobility as the standard BP specimen was confirmed. Inthe TLC analysis, it was found that, when 20 μl of the culturesupernatant oxidized with potassium iodide as mentioned in Example 5were analyzed, 1.37 μg of BP were produced per 20 μl of the culturesupernatant upon calculation from the ultraviolet absorption intensityof the spot of BP and that value is identical with the value calculatedfrom the result of the HPLC analysis which was 0.76 μg per ml of theculture supernatant.

Example 6 Preparation of Escherichia coli Producing the BiopterinCompound

[0208] In order to prepare a host bacterium being able to produce GTPwhich is a precursor of THBP in more quantities than the wild type, aregulatory mutant for synthesis of purine was prepared as follows.

[0209] Firstly, in an M9 minimum agar medium containing 20, 50, 100 and500 μg/ml of 8-azaguanine and decoynine, sensitivity of those substancesto Escherichia coli JM101 was tested and it became clear that thesensitivity was available to 100 μg/ml or more 8-azaguanine and to 500μg/ml or more decoynine.

[0210] Composition of the M9 minimum agar medium: 2 g/L of glucose, 6g/L of Na₂HPO₄, 3 g/L of KH₂PO₄, 0.5 g/L of NaCl, 1 g/L of NH₄Cl, 2 mMof MgSO₄, 0.1 mM of CaCl₂ and 15 g/L of agar; pH 7.4.

[0211] Then, Escherichia coli JM101 which was mutated byN-methyl-N-nitro-nitrosoguanidine (Miller, 1972, Experiments inMolecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) was sowed on a plate of the M9 minimum agar medium containing 100μg/ml of 8-azaguanine or 500 μg/ml of decoynine and incubated at 37° C.to give a resistant mutant. From each of the resistant mutant strains,50 strains each were selected and expression plasmid pSTV28-GPS wasintroduced thereinto by a calcium chloride method. The strain preparedas such was cultured for 48 hours in the above-mentioned NUCA medium andthe amount of the biopterin compound produced by each strain wascompared.

[0212] As a result, among the 8-azaguanine-resistant strains, there wasobtained an AG14/pSTV28-GPS strain having a productivity of thebiopterin compound of about 10-fold as compared with the parent strain(JM101/pSTV28-GPS). Thus, it became clear that, when 8-azaguanineresistance is given to Escherichia coli, a strain where the productivityof the biopterin compound increased was able to be prepared (Table 1).Incidentally, it became clear that, with regard to this strain, DHBP wasdetected in the culture broth in addition to BP and that a part of theproduced THBP was naturally oxidized being present as DHBP or BP (FIG.17).

Example 7 Culture of Escherichia coli Producing the Biopterin CompoundUsing a Jar Fermenter and Purification of BP

[0213] A bacterium AG14/pSTV28-GPS strain being able to produce thebiopterin compound was cultured using a 3-liter jar fermenter. As amedium for the jar fermenter, the above-mentioned NUCA medium containing0.5 mM of IPTG was used. Firstly, the AG14/pSTV28-GPS strain wascultured in an LB medium for one night, 10 ml thereof were inoculated on2 L of the NUCA medium containing 0.5 mM of IPTG and cultured at 37° C.while the level of dissolved oxygen was kept at 30%. After 2% ofglycerol added in the initial stage of the culture was consumed, 80%glycerol was added successively and the cultivation was carried out for48 hours.

[0214] An HPLC analysis was carried out for a part of the culture broth.Thus, the produced THBP was converted by oxidizing with potassium iodidesolution as same as in Example 4 and analysis was conducted by means offluorescent analysis using an HPLC. The HPLC was carried out as same asin Example 5. The result was that, when determined from the producedamount of BP calculated from the peak area, it was found that 350 mg/Lof THBP was produced by the culture for 48 hours.

[0215] In order to obtain the BP from the resulting culture broth, 200ml of the above culture broth were centrifuged to remove the cells andone-tenth of potassium iodide solution (1N hydrochloric acid solutioncontaining 0.9% of 12 and 1.8% of KI) was added to the supernatant so asto oxidize to BP. After that, the mixture was allowed to stand for 1hour shielding the light and adjusted to pH 7.0 by adding 5M aqueoussolution of sodium hydroxide thereto. Then the solution was cooled withice so that BP was separated out and precipitated, the resultingprecipitate was recovered by centrifugation, pure water was addedthereto and the mixture was adjusted to pH 2 with hydrochloric acidwhereby the precipitate was dissolved. This solution was charged to acolumn of Dowex 1×8 (10 mm×100 mm), washed with 20 ml of pure water andeluted with 0.5 M NaCl at a flow rate of 1 ml/minute. Every 2 ml of theresulting eluate were collected and the 8th to the 16th fractions werepooled. One half of the pooled fractions was charged to a column (10mm×100 mm) of Florisil equilibrated with 0.5 M formic acid, washed with20 ml of 0.5 M formic acid and eluted with 2N HCl. Every 1.8 ml of theresulting eluate were collected and the 4th to the 16th fractions werepooled whereby 2 mg of BP of 98% purity were obtained.

Example 8 Optimization of Culture Condition and Increase in theProducing Amount of the Biopterin Compound

[0216] In the culture using the three-liter jar fermenter mentioned inExample 17, culture was carried out where incubating temperature, pH ofmedium and level of dissolved oxygen were controlled to predeterminedones. Those conditions were as follows.

[0217] (1) Cultivation temperature is 37° C.

[0218] (2) ph value is 6.5.

[0219] (3) Dissolved oxygen level is 30%.

[0220] pH of the medium was controlled by addition of 28% aqueousammonia, while dissolved oxygen level was controlled by an increase ofrevolution upon stirring. With regard to glycerol which is a carbonsource, 2% were added at the start of the culture and, afterconsumption, 80% glycerol were continuously added at the flow rate of 10ml/hour. As a result, it was found that 2 g/L of THBP were produced uponculture for 48 hours when determined from the produced amount of BPcalculated from the peak area of the HPLC analysis (FIG. 18).

Example 9 Increase in the Produced Amount of the Biopterin Compound byIncreased Expression of guaBA Gene

[0221] Cloning of guaBA gene (Tiedeman, A. A., et al., J. Biol. Chem.260:8676-8679, 1985 and Nucleic Acids Res. 13:1303-1316, 1985 [GenBankM10101]) was carried out from Escherichia coli (W 3110 strain) genomicDNA by a PCR. Escherichia coli (W 3110 strain) genomic DNA was preparedby a reported method (Seibutsu Kogaku Jikkensho [published by Baifukan,p. 97-98]). A PCR was carried by a conventional manner using senseprimer P 33 (SEQ ID NO: 33) and antisense primer P 34 (SEQ ID NO: 34) toprepare DNA containing Escherichia coli guaBA gene. Then the resultingguaBA gene was subcloned to vector pCR2.1 (Invitrogen) to preparepCR2.1-guaBA. After that, pCR2.1-guaBA was cleaved by BamHI and XhoI anda fragment of 3 kb containing guaBA gene was inserted into BamHI andXhoI fragments (3.9 kb) of pMW218 (Nippon Gene) to prepare pMW218-guaBA(FIG. 19). This plasmid was introduced into AG14/pSTV28-GPS strain by acalcium chloride method to prepare AG14/(pSTV28-GPS, pMW218-guaBA).Selection and culture of the transformed strain were carried out in amedium containing kanamycin (25 μg/ml) and chloramphenicol (25 μl/ml).The prepared strain was cultured by a method mentioned in Example 4 andthe produced amount was measured from the peak area by an HPLC analysiswhereupon 580 mg/L of BP were found to be produced (Table 1).

Example 10 Increase in Produced Amount of the Biopterin Compound due toIncrease in Expressed Amount of GCH

[0222] pSTV28-GCH and pUC18-PTPS were treated with EcoRI and SalI toisolate folE gene and rat PTPS gene and each of them was inserted intoEcoRI and SalI sites existing in a multi-cloning sites of pTWV228(Takara Shuzo) to prepare pTWV228-GCH and pTWV228-PTPS. Similarly,pUC19-SPR was treated with HindIII to isolate rat SPR gene and insertedinto HindIII existing in a multi-cloning site of pTWV229 (Takara Shuzo)to prepare pTWV229-SPR. Each of the three plasmids prepared as such wasintroduced into AG14/(pSTV28-GPS, pMW218-guaBA) strain and producedamount of the biopterin compound was measured by the method mentioned inExample 4. Selection and culture of the transformed strain were carriedout in a medium containing ampicillin (25 μg/ml), kanamycin (25 μg/ml)and chloramphenicol (25 μg/ml).

[0223] Produced amounts of the prepared bacteria were 524 mg/L forAG14/(pSTV28-GPS, pMW218-guaBA, pTWV228-GCH), 337 mg/L forAG14/(pSTV28-GPS, pMW218-guaBA, pTWV228-PTPS) and 465 mg/L forAG14/(pSTV28-GPS, pMW218-guaBA, pTWV229-SPR). As a result that theplasmid was newly introduced, an increase in the expressed amount ofeach enzyme was confirmed upon analysis by SDS-PAGE.

[0224] From the above result, it was found that, with regard to thethree kinds of enzymatic genes participating in the THBP production,produced amount of the biopterin compound was able to be improved whenits expressed amount was increased and that, particularly when theexpressed amount of GCH gene was increased, produced amount of biopterincompound was able to be further improved.

Example 11 Increase in Production of the Biopterin Compound byUtilization of Bacillus subtilis GCH Gene (mtrA)

[0225] Cloning of mtrA (Gollnick, P., et al. Proc. Natl. Acad. Sci. U.S.A. 87:8726-8730, 1990, [GenBank M37320]) which was a Bacillus subtilisGCH gene was carried out from Bacillus subtilis lAl strain by a PCR. TheBacillus subtilis genomic DNA was prepared by a reported method (IdenshiHatsugen Jikken Manual [Kodansha Scientific, p. 31-33]). A PCR wascarried out by a conventional method using sense primer P35 (SEQ ID NO:35) and antisense primer P 36 (SEQ ID NO: 36) to prepare the DNAcontaining Bacillus subtilis mtrA gene. After that, the resulting PCRproduct was used as a template and a PCR was carried out once againusing sense primer P37 (SEQ ID NO: 37) and antisense primer P 38 (SEQ IDNO: 38) and the product was cleaved by EcoRI and XbaI and connected to adigested product of pUC18 by EcoRI-XbaI to prepare pUC18-mtrA.

[0226] After that, mtrA gene was excised from pUC18-mtrA by EcoRI andXbaI and they were connected to a fragment made by digestion ofpSTV28-GPS with EcoRI and SphI (3.0 kb) and a fragment made by digestionof pSTV28-GPS with XbaI and SphI (1.4 kb) to prepare pSTV28-MPS (FIG.20). Then AG14/pMW218-guaBA strain was transformed by the resultingpSTV28-MPS to prepare AG14/(pSTV28-MPS, pMW218-guaBA) and the producedamount of THBP was measured by the method mentioned in Example 4.Cultivation was carried out in a medium containing kanamycin (25 μg/ml)and chloramphenicol (25 μg/ml). As a result, the strain[AG14/(pSTV28-MPS, pMW218-guaBA)] having the GCH gene of Bacillussubtilis showed high produced amount of THBP (770 mg/L) and it was foundthat, when the GCH gene (mtrA) of Bacillus subtilis was used, producedamount of the biopterin compound further increased (Table 1). TABLE 1THBP Strain Production [Detected as Bp] JM101/pSTV28-GPS  23 mg/LAG14/pSTV28-GPS 250 mg/L AG14/(pSTV28-GPS, pMW218-guaBA) 580 mg/LAG14/(pSTV28-MPS, pMW218-guaBa) 770 mg/L

Example 12 Culture of Strain with Increased guaBA Expression and StrainCarrying GCH Homologue Gene Derived from Bacillus subtilis by JarFermenter

[0227] AG14/(pSTV28-GPS, pMW218-guaBA) which was a strain withincreasedguaBAexpression andAG14/(pSTV28-MPS, pMW218-guaBA), in whichGCH gene from Bacillus subtilis was introduced, were subjected to atwo-liter scale cultivation according to a method mentioned in Examples7 and 8. As a result, BP calculated from the peak area in the HPLCanalysis was produced in an amount of 2.4 g/L in the case ofAG14/(pSTV28-GPS, pMW218-guaBA) and in an amount of 4 g/L in the case ofAG14/(pSTV28-MPS, pMW218-guaBA) by a culture for 42 hours (FIG. 21).

Example 13 Preparation of Expression Vectors pDG148 MPS and pDG148 MPSAIfor Bacillus subtilis

[0228] Expression vectors pDG148 MPS and pDG148 MPSAI for Bacillussubtilis were prepared as follows.

[0229] (1) Preparation of pUC18SD (Refer to FIG. 22)

[0230] Oligonucleotides P39 (SEQ ID NO: 39) and P40 (SEQ ID NO: 40)having complementary sequences were mixed and, on the other hand, P41(SEQ ID NO: 41) and P42 (SEQ ID NO: 42) having the complementarysequences were mixed to prepare two double stranded DNA fragments. Eachfragment was subjected to a reaction of adding a phosphate group to each5′terminus by T4 polynucleotide kinase and cloned to the site ofHindIII-EcoRI of pUC18.

[0231] The sequence of about 45 bp between HindIII and EcoRI of theresulting plasmid pUC18SD contained a DNA sequence coding for thetranslation initiation region starting from SD sequence (Shine-Dalgarnosequence) suitable for gene expression of Bacillus subtilis and aminoacid sequence of MSNITNS(methionine-serine-asparagine-isoleucine-threonine-asparagi ne-serine)(Fujita, et al. Microbiology, 140:6571-6580, 1998).

[0232] (2) Preparation of pUC18AE (Refer to FIG. 23)

[0233] pUC18 was completely digested by EcoRI, Mung Bean Nuclease wasfurther added thereto, the mixture was incubated at 37° C. for 15minutes, purification with ethanol was conducted and a ligation reactionwas carried out to prepare pUC18AE. The resulting plasmid was confirmednot to be cleaved by EcoRI. By a DNA sequencing, it was confirmed thatthe recognition sequence 5′-GAATTC-3′ of EcoRI was changed to5′-GATT-3′.

[0234] (3) Preparation of pUC18AESDmtrA (Refer to FIG. 24)

[0235] For cloning of the mtrA gene, a PCR was carried out usingBacillus subtilis genomic DNA as a template and using P43 (SEQ ID NO:43) and P44 (SEQ ID NO:44), then a PCR was carried out using theamplified DNA as a template and using P45 (SEQ ID NO: 45) and P46 (SEQID NO: 46), P45 (SEQ ID NO: 45) and P47 (SEQ ID NO: 47) to introducecleavage sites for restriction enzymes. Termini of the resulting DNAfragments were digested by EcoRI and PstI and connected to HindIII-EcoRIDNA fragment (SD sequence) prepared from pUC18SD and 2.6 kb HindIII-PstIDNA fragment derived from pUC18AE to prepare pUC18AESDmtrA. FIG. 31shows an amino acid sequence corresponding to a DNA base sequence ofmtrA cloned on pUC18AESDmtrA. Incidentally, the underlined part shows anamino acid sequence derived from CcpA protein to be added to aminoterminus of mtrA (Fujita, et al., Microbiology, 140:6571-6580, 1998).

[0236] (4) Preparation of pUC18SDPTPS (Refer to FIG. 25)

[0237] A PCR was carried out using pUC18SD as a template and usingprimers P50 (SEQ ID NO: 50) and P48 (SEQ ID NO: 48) and termini of theresulting DNA fragment were digested by XbaI and EcoRI to prepare a DNAfragment containing SD sequence. Similarly, pUC18-PTPS prepared in thestep of Example 1 as shown by FIG. 4 was used as a template and primersP50 (SEQ ID NO: 50) and P49 (SEQ ID NO: 49) were used to prepare a 0.45kb EcoRI-SalI fragment containing PTPS gene. They were connected to a2.6 kb XbaI-SalI DNA fragment derived from pUC18 to prepare pUC18SDPTPS.FIG. 32 shows an amino acid sequence corresponding to a DNA basesequence of PTPS cloned on pUC18SDPTPS. Incidentally, the underlinedpart shows an amino acid sequence derived from CcpA protein to be addedto amino terminus of PTPS (Fujita, et al., Microbiology, 140:6571-6580,1998).

[0238] (5) Preparation of pUC18AESDSPR (Refer to FIG. 26)

[0239] A PCR was carried out using pUC18SD as a template and usingprimers P51 (SEQ ID NO: 51) and P53 (SEQ ID NO: 53) and termini of theresulting DNA fragment were digested by SailI and EcoRI to prepare a DNAfragment containing SD sequence. Similarly, pUCl9-SPR prepared in thestep of Example 1 as shown by FIG. 5 was used as a template and primersP52 (SEQ ID NO: 52) and P53 (SEQ ID NO: 53) were used to prepare a 0.8kb EcoRI-SphI fragment containing SPR gene. They were connected to a 2.6kb SalI-SphI DNA fragment derived from pUC18AE to prepare pUC18AESDSPR.FIG. 33 shows an amino acid sequence corresponding to a DNA basesequence of SPR cloned on pUC18AESDSPR. Incidentally, the underlinedpart shows an amino acid sequence derived from CcpA protein to be addedto amino terminus of SPR (Fujita, et al., Microbiology, 140:6571-6580,1998).

[0240] (6) Preparation of pSL1180PS (refer to FIG. 27) pUC18SDPTPSprepared by the step shown in FIG. 25 as above was digested by XbaI andSailI to prepare a fragment (SD sequence+PTPS) of 0.5 kb. After that,pUC18AESDSPR prepared by the step shown in FIG. 6 as above was digestedby SalI and SphI to purify a fragment (SD sequence+SPR) of 0.85 kb.Those fragments were connected to XbaI- and SphI-digested product (3.4kb) of pSL1180 to prepare pSL1180PS.

[0241] (7) Preparation of pSL1180 MPS (Refer to FIG. 28)

[0242] pUC18AESDmtrA prepared by the step shown in FIG. 24 as above wasdigested by HindIII and XbaI to prepare a 0.63 kb fragment and that wasconnected to a 4.7 kb fragment obtained by digestion of pSL1180PS byHindIII and XbaI to prepare pSL1180 MPS.

[0243] (8) Preparation of pDG148 MPS, Expression Vector for Bacillussubtilis (refer to FIG. 29)

[0244] pSL1180 MPS prepared by the step shown in FIG. 28 as above wasdigested by HindIII and SphI to prepare a DNA fragment of 2.0 kb wheremtrA, PTPS and SPR genes were aligned successively. On the other hand, ashuttle vector pDG148 which was able to be used for Escherichia coli andBacillus subtilis (Karmazyn-Campelli, et al., Cell, 52, 697-704, 1988)was digested by HindIII and SphI to prepare a DNA fragment of 8.2 kb andthat was connected to the previously prepared DNA fragment of 2.0 kb toprepare pDG148 MPS, an expression vector for Bacillus subtilis.Incidentally, in FIG. 29, Pspac is spac promoter, lacI is lacI gene,lacO is expression regulatory region by lacI protein, Km iskanamycin-resistant gene, Ap is ampicillin-resistant gene and ori isorigin of replication of plasmid.

[0245] In this expression vector pDG148 MPS, a lacO sequence is coded atthe downstream of spac promoter (Pspac) and lacI gene which induces theexpression in Bacillus subtilis is coded at the downstream ofmulti-cloning site. Accordingly, in Bacillus subtilis, transcription byspacpromoter is suppressed by lacI protein. However, when IPTG which isan inducing substance is added to the medium, transcription by spacpromoter is activated and expression of gene at the downstream isinduced. At the upper stream of mtrA, PTPS and SPR genes, there arealigned SD sequence each being suitable for gene expression of Bacillussubtilis and DNA sequence coding for the translation initiation regionstarting from an amino acid sequence of MSNITNS(methionine-serine-asparagine-isoleucine-threonine-asparagine-serine).Transcription of those three genes aligned in a form of operon isinduced by spac promoter.

[0246] (9) Preparation of pDG148 MPSAI, Expression Vector for Bacillussubtilis (refer to FIG. 30)

[0247] The pSL1180 MPS prepared by the step shown in FIG. 28 as abovewas digested by HindIII and BamHI which were restriction enzymes toprepare a DNA fragment of 2.0 kb where mtrA, PTPS and SPR genes werealigned successively. This was connected to the above digested product(6.9 kb) of pDG148 by HindIII and BamHI to prepare pDG148 MPSAI, anexpression vector for Bacillus subtilis. Incidentally, in FIG. 30, Pspacis spac promoter, laco is expression regulatory region by lacI protein,Km is kanamycin-resistant gene, Ap is ampicillin-resistant gene and oriis origin of replication of plasmid.

[0248] In this expression vector pDG148 MPSAI, a lacO sequence isavailable at the downstream of spac promoter (Pspac) but, since lacIgene part is deficient from pDG148, no suppression to spac promoter isresulted in Bacillus subtilis. Accordingly, expression of gene atdownstream is constantly induced. At the upper stream of mtrA, PTPS andSPR genes, there are aligned SD sequence each being suitable for geneexpression of Bacillus subtilis and DNA sequence coding for thetranslation initiation region starting from an amino acid sequence ofMSNITNS. Transcription of those three genes aligned in a form of operonis induced by spac promoter.

Example 14 Production of the Biopterin Compound by Bacillus subtilis

[0249]Bacillus subtilis lAl strain (trpC2) (Fujita, et al.,Microbiology, 140:6571-6580, 1998) was transformed by pDG148 MPS orpDG148 MPSAI according to the following method. Thus, each of theplasmids of pDG148 MPS and pDG148 MPSAI used for the transformation wasa vector derived from a shuttle vector pDG148 (Karmazyn-Campelli, etal., Cell, 52, 697-704, 1988) which was transformable for bothEscherichia coli and Bacillus subtilis and, in the case of Escherichiacoli, resistance to ampicillin was able to be used as a selective markerwhile, in the case of Bacillus subtilis, resistance to kanamycin wasable to be used as a selective marker.

[0250]Bacillus subtilis lAl strain was streaked on a TBABG plate and apre-incubation was carried out for 12 hours. [Composition in 1L of theTBABG plate: 10 g of tryptone, 3 g of beef extract, 5 g of NaCl, 15 g ofagar powder and 1.8 g of glucose]

[0251] Cells were scraped off from the plate and suspended in 2 ml of aCI medium. [Composition of the CI medium: 0.2% of (NH₄)₂SO₄, 1.4% ofK₂HPO₄, 0.6% of KH₂PO₄, 0.1% of Na₃ citrate.2H₂O, 5 mM of MgSO₄, 0.5% ofglucose, 0.02% of Casamino acid and 50 μg/ml of tryptophane (trp)]

[0252] The resulting suspension was diluted with the CI medium to as tomake OD₆₀₀ mm=0.08 and subjected to a shaking culture at 37° C. Duringthe cultivation, values of OD_(600 nm) were appropriately measured toconfirm that the growth entered the stationary phase.

[0253] The culture broth (2.5 ml) was centrifuged (at 2500 rpm and roomtemperature (RT) for 10 minutes) and the collected cells were suspendedin 5 ml of a CII medium and subjected to a shaking culture for about 30minutes to give competent cells. [Composition of the CII medium: 0.2% of(NH₄)₂SO₄, 1.4% of K₂HPO₄, 0.6% of KH₂PO₄, 0.1% of Na₃ citrate.2H₂O, 5MM of MgSO₄, 0.5% of glucose, 0.01% of Casamino acid and 5 μg/ml oftryptophane (trp)]

[0254] The competent cell (1 ml) was mixed with 1 μg of pDG148 MPS orpDG148 MPSAI prepared from Escherichia coli JM101 strain which was arecA⁺ strain and subjected to a shaking culture gently at 37° C. for 2hours. After that, the culture broth was streaked to a TBABG platecontaining 5 μg/ml of kanamycin and incubated at 37° C. for about 12hours to give each of the transformants 1A1/pDG148 MPS and 1A1/pDG148MPSΔI.

[0255] Colony of each of the resulting transformants was pre-cultured in3 ml of an LB medium containing 5 μg/ml of kanamycin (Km) for about 3hours. After that, it was inoculated on an NU medium (3.5 ml) containing2% of glucose, 1 mM of IPTG and 5 μg/ml of Km and subjected to a shakingculture at 37° C. for 20 hours. Similar cultivation was carried out in amedium containing no kanamycin for Bacillus subtilis as a control foreach strain as well. [Composition of the NU medium (per 1L): 4 g ofyeast extract, 4 g of KH₂PO₄, 4 g of K₂HPO₄, 2.8 g of Na₂HPO₄, 0.2 g ofNH₄Cl, 2 g of MgSO₄₀.7H₂O, 0.04 g of FeSO₄, 0.04 g of CaCl₂₀.2H₂O, 0.01g of MnSO₄₀.5H₂O, 0.01 g of AlCl₃₀.6H₂O, 0.004 g of COCl₂₀.6H₂O, 0.002 gof ZnSO₄₀.7H₂O, 0.002 g of Na₂MoO₄₀.2H₂O, 0.001 g of CuCl₂₀.2H₂O and0.0005 g of H₃BO3]

[0256] The result was that, in all strains including the control strain,values of OD_(600 nm) were 10 to 12. Growth of the cells was good andany changes by transformation or gene expression induced by IPTG werenot observed. The supernatant of culture broth after 20 hours from thestart of the culture was oxidized by a method mentioned in Example 4 andsubjected to an HPLC analysis.

[0257] Result of the HPLC analysis is shown in FIG. 34. In the 1A1strain (right end) where no plasmid was introduced, no peak was noted atthe eluted position of the standard BP specimen while, in other twostrains (1A1/pDG148 MPS and 1A1/pDG148 MPSΔI), peaks were noted at theeluting position of the standard BP specimen whereby it became clearthat the prepared Bacillus subtilis hadaBP-producingability.Uponcalculationfromthe area ratio of the peak areas, amount of theproduced BP was about 0.45 μg/ml in all cases.

INDUSTRIAL APPLICABILITY

[0258] The present invention achieves an advantage that the biopterincompounds being expected to have pharmacological effect can be producedin large quantities in an industrial scale starting from less expensivemedium materials. As a result, studies for the biopterin compounds,particularly for pharmacological action thereof, are now able to beeasily carried out and development of new pharmaceuticals is able to bepromoted.

[0259] The present invention also achieves an advantage that productionof DHBP and BP which are oxidized products of THBP becomes possible. Asa result, studies for metabolism of biopterin compounds are now able tobe easily carried out.

1 53 1 21 DNA Artificial sequence Primer P1 1 aaatcataaa tgccatcact c 212 21 DNA Artificial sequence Primer P2 2 gccttttaat cagttgtgat g 21 3 25DNA Artificial sequence Primer P3 3 aaagaattca tgccatcact cagta 25 4 23DNA Artificial sequence Primer P4 4 gccttttaac tagttgtgat gac 23 5 26DNA Artificial sequence Primer P5 5 ccggaattcc atgccatcac tcagta 26 6 31DNA Artificial sequence Primer P6 6 gatttgtcga ctatcaggct gaaaatcttc t31 7 21 DNA Artificial sequence Primer P7 7 cttgtgggtc tttggtctga a 21 821 DNA Artificial sequence Primer P8 8 gggtaggtga tgactgctgt g 21 9 24DNA Artificial sequence Primer P9 9 tttggtctga attccatgaa cgcg 24 10 33DNA Artificial sequence Primer P10 10 tattaaaact agtatctatt ctcctttgtagac 33 11 30 DNA Artificial sequence Primer P11 11 gcccaagctt gtttgacagcttatcatcga 30 12 31 DNA Artificial sequence Primer P12 12 gatttgtcgactatcaggct gaaaatcttc t 31 13 21 DNA Artificial sequence Primer P13 13ggcaggctag gttgcgctgt c 21 14 21 DNA Artificial sequence Primer P14 14gggcttaaat gtcatagaag t 21 15 30 DNA Artificial sequence Primer P15 15aaaggatccg gcaggctagg ttgcgctgtc 30 16 26 DNA Artificial sequence PrimerP16 16 cctgactagt taaatgtcat agaagt 26 17 30 DNA Artificial sequencePrimer P17 17 atgaagcttg ggcaggctag gttgcgctgt 30 18 31 DNA Artificialsequence Primer P18 18 gatttgtcga ctatcaggct gaaaatcttc t 31 19 30 DNAArtificial sequence Primer P19 19 gagcgtcgac tgagggcaac gcaattaatg 30 2030 DNA Artificial sequence Primer P20 20 cgcgggatcc ctattctcctttgtagacca 30 21 30 DNA Artificial sequence Primer P21 21 gagcggatcctgagcgcaac gcaattaatg 30 22 30 DNA Artificial sequence Primer P22 22cgcggcatgc ttaaatgtca tagaagtcca 30 23 37 DNA Artificial sequence PrimerP23 23 ttcaaggatc ccaaaatgca taacatccaa ttagtgc 37 24 38 DNA Artificialsequence Primer P24 24 ctattactcg agttaataca tacacgatat atcgtcgc 38 2529 DNA Artificial sequence Primer P25 25 gattggatcc accatgaacg cggcggttg29 26 29 DNA Artificial sequence Primer P26 26 gcgactcgag tctattctcctttgtagac 29 27 43 DNA Artificial sequence Primer P27 27 gaccggatccaccatggaag gtggcaggct aggttgcgct gtc 43 28 31 DNA Artificial sequencePrimer P28 28 ccgcctcgag ttaaatgtca tagaagtcca c 31 29 25 DNA Artificialsequence Primer P29 29 atccgcatgc acggattaga agccg 25 30 29 DNAArtificial sequence Primer P30 30 atgtactagt ctgcgttatc ccctgattc 29 3125 DNA Artificial sequence Primer P31 31 atccactagt acggattaga agccg 2532 29 DNA Artificial sequence Primer P32 32 atgtactagt ctgcgttatcccctgattc 29 33 23 DNA Artificial sequence Primer P33 33 gtaaagtaccagtgaccgga agc 23 34 27 DNA Artificial sequence Primer P34 34 cagctggtttaattaatcga tgttagt 27 35 23 DNA Artificial sequence Primer P35 35cagggcattc actttgcttt tag 23 36 23 DNA Artificial sequence Primer P36 36ttcgcttacg caggtatcat tat 23 37 30 DNA Artificial sequence Primer P37 37attacgaatt ccatgaaaga agttaataaa 30 38 30 DNA Artificial sequence PrimerP38 38 atgcctgcag tctagacgca ttagtcctgg 30 39 30 DNA Artificial sequencePrimer P39 39 agcttgtgta tccagtaaaa ggagtggttt 30 40 30 DNA Artificialsequence Primer P40 40 cctaaaacca ctccttttac tggatacaca 30 41 19 DNAArtificial sequence Primer P41 41 taggatgacg aatattacg 19 42 19 DNAArtificial sequence Primer P42 42 aattcgtaat attgctcat 19 43 23 DNAArtificial sequence Primer P43 43 cagggcattc actttgcttt tag 23 44 23 DNAArtificial sequence Primer P44 44 ttcgcttacg caggtatcat tat 23 45 30 DNAArtificial sequence Primer P45 45 attacgaatt ccatgaaaga agttaataaa 30 4630 DNA Artificial sequence Primer P46 46 gctctagacg cattagtcctggcgtttaat 30 47 30 DNA Artificial sequence Primer P47 47 atgcctgcagtctagacgca ttagtcctgg 30 48 30 DNA Artificial sequence Primer P48 48gtgcctctag agtgtatcca gtaaaaggag 30 49 30 DNA Artificial sequence PrimerP49 49 caggcgtcga cttcgaaagc ggccgcgact 30 50 24 DNA Artificial sequencePrimer P50 50 ctcattaggc accccaggct ttac 24 51 30 DNA Artificialsequence Primer P51 51 gtgccgtcga cgtgtatcca gtaaaaggag 30 52 30 DNAArtificial sequence Primer P52 52 caggcgcatg cttcgaaagc ggccgcgact 30 5320 DNA Artificial sequence Primer P53 53 gcaacgcaat taatgtgagt 20

1. In a process for production of a biopterin compound by a transformedcell, a process for production of the biopterin compound which ischaracterized in that (a) a host cell is transformed by at least onegene of enzyme participating in biosynthesis of tetrahydrobiopterin, (b)the resulting transformed cell is cultured to producetetrahydrobiopterin, (c) the resulting tetrahydrobiopterin is oxidizedif necessary and (d) one or more biopterin compound(s) selected from theresulting tetrahydrobiopterin and dihydrobiopterin and biopterin wherethe said tetrahydrobiopterin is oxidized is/are collected.
 2. Theprocess for production of the biopterin compound according to claim 1,wherein the biopterin compound in the culture broth or in a processedproduct thereof is oxidized and biopterin is collected therefrom.
 3. Theprocess for production of the biopterin compound according to claim 1 or2, wherein the collected dihydrobiopterin and/or biopterin are/isreduced to produce tetrahydrobiopterin.
 4. The process for production ofthe biopterin compound according to claims 1 to 3, wherein the enzyme(s)participating in the biosynthesis of tetrahydrobiopterin is/are one tothree kind(s) of enzyme(s) selected from a group consisting of GTPcyclohydrase 1,6-pyruvoyltetrahydropterin synthase and sepiapterinreductase.
 5. The process for production of the biopterin compoundaccording to claims 1 to 3, wherein the transformation is carried out byan expression vector having gene of 6-pyruvoyltetrahydropterin synthaseand gene of sepiapterin reductase.
 6. The process for production of thebiopterin compound according to claim 5, wherein the host cell is amutant cell having a GTP cyclohydrase I activity which is not less thanthe GTP cyclohydrase I activity inherent to the cell of a wild type. 7.The process for production of the biopterin compound according to claim4, wherein the GTP cyclohydrase I gene to be introduced into the hostcell is mtrA gene derived from Bacillus subtilis.
 8. The process forproduction of the biopterin compound according to claims 1 to 7, whereinthe host cell is a prokaryotic cell.
 9. The process for production ofthe biopterin compound according to claim 8, wherein the prokaryote isEscherichia coli, Bacillus subtilis or Actinomyces.
 10. The process forproduction of the biopterin compound according to claims 1 to 7, whereinthe host cell is an eukaryotic cell.
 11. The process for production ofthe biopterin compound according to claim 10, wherein the eukaryote isyeast or filamentous fungi.
 12. The process for production of thebiopterin compound according to claim 11, wherein the yeast is methanolassimilating yeast or fission yeast.
 13. The process for production ofthe biopterin compound according to claim 11, wherein the yeast isSaccharomyces yeast.
 14. The process for production of the biopterincompound according to claims 1 to 13, wherein the host cell is a mutantcell having a GTP synthesizing ability of not less than the GTPsynthesizing ability of the cell of a wild type.
 15. The process forproduction of the biopterin compound according to claim 14, wherein thehost cell is a mutant cell having a 8-azaguanine resistance of not lessthan the 8-azaguanine resistance of the cell of a wild type.
 16. Theprocess for production of the biopterin compound according to claim 14or 15, wherein the host cell is a genetically recombinant cell whereguaBA gene coding for IMP dehydrogenase and GMP synthase is introducedthereinto.
 17. A transformed cell used for the production of thebiopterin compound mentioned in claims 1 to 16 in which gene of anenzyme participating in biosynthesis of tetrahydrobiopterin isintroduced into a host cell.
 18. The transformed cell according to claim17, wherein the host is a mutant cell having a GTP synthesizing abilityof not less than the GTP synthesizing ability of the cell of a wildtype.
 19. The transformed cell according to claim 18, wherein the hostcell is a mutant cell having a 8-azaguanine resistance of not less thanthe 8-azaguanine resistance of the cell of a wild type.
 20. Thetransformed cell according to claim 18 to 19, wherein the host cell is agenetically recombinant cell where guaBA gene coding for IMPdehydrogenase and GMP synthase is introduced thereinto.
 21. Thetransformed cell according to claim 18, wherein the host cell is amutant cell having a GTP cyclohydrase I activity which is not less thanthe GTP cyclohydrase I activity inherent to the cell of a wild type andgene of 6-pyruvoyltetrahydropterin synthase and gene of sepiapterinreductase are introduced into the said host cell.
 22. The transformedcell according to claims 17 to 21, wherein the GTP cyclohydrase I geneto be introduced into the host cell is mtrA gene derived from Bacillussubtilis.